Adjuvanting meningococcal factor h binding protein

ABSTRACT

Factor H binding protein (fHBP) has been proposed for use in immunising against serogroup B meningococcus (‘MenB’). This antigen can be efficiently adsorbed to an aluminium hydroxyphosphate adjuvant by (i) ensuring that adsorption takes place at a pH which is equal to or below the adjuvant&#39;s point of zero charge (PZC), and/or (ii) selecting a fHBP and adjuvant with an isoelectric point/PZC within the range of 5.0 to 7, and/or (iii) selecting a fHBP with an isoelectric point above the adjuvant&#39;s PZC and using a buffer to bring the pH to within 1.2 pH units of the PZC. The adsorption is particularly useful for compositions which include multiple fHBP variants, and also in situations where an aluminium hydroxide adjuvant should be avoided. Buffered pharmaceutical compositions can include at least two different meningococcal fHBP antigens, both of which are at least 85% adsorbed to aluminium hydroxyphosphate adjuvant.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.15/393,092, filed Dec. 28, 2016, which is a Divisional of U.S. patentapplication Ser. No. 14/307,001, filed Jun. 17, 2014, now U.S. Pat. No.9,572,884; which is a Continuation of U.S. patent application Ser. No.13/767,853, filed Feb. 14, 2013, now U.S. Pat. No. 8,834,888; which is aContinuation of U.S. patent application Ser. No. 13/403,865, filed Feb.23, 2012, now U.S. Pat. No. 8,398,988; which is a Continuation of U.S.patent application Ser. No. 13/260,534, claiming an international filingdate of Mar. 24, 2010, now Abandoned; which is the National Stage ofInternational Patent Application No. PCT/IB2010/000733, filed Mar. 24,2010; which claims the benefit of U.S. Provisional Patent ApplicationNo. 61/162,999, filed Mar. 24, 2009, each of which is herebyincorporated by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 303822003104SeqList.txt,date recorded: May 24, 2017, size: 74 KB).

TECHNICAL FIELD

This invention is in the field of meningococcal vaccines, in particularthose containing fHBP antigen.

BACKGROUND ART

Neisseria meningitidis (meningococcus) is a Gram-negative sphericalbacterium. Current meningococcal vaccines are also based on capsularsaccharides. These include monovalent serogroup C conjugate vaccines and4-valent conjugate mixtures for serogroups A, C, W135 and Y. There iscurrently no useful vaccine authorised for general use against serogroupB (‘MenB’).

One antigen which has been proposed for use in immunising against MenBis the factor H binding protein (fHBP). This antigen has also beencalled protein ‘741’ (SEQ IDs 2535 & 2536 in ref. 34), ‘NMB1870’,‘GNA1870’ [refs. 1-3], ‘P2086’, ‘LP2086’ or ‘ORF2086’ [4-6]. The proteinhas been well studied. It is naturally a lipoprotein and is expressedacross all meningococcal serogroups. The structure of fHbp's C-terminalimmunodominant domain (‘fHbpC’) has been determined by NMR [7]. Thispart of the protein forms an eight-stranded β-barrel, whose strands areconnected by loops of variable lengths. The barrel is preceded by ashort α-helix and by a flexible N-terminal tail.

The fHBP antigen falls into three distinct variants [8] and it has beenfound that serum raised against a given family is bactericidal withinthe same family, but is not active against strains which express one ofthe other two families i.e. there is intra-family cross-protection, butnot inter-family cross-protection. Thus reference 8 proposes to combinedifferent variants of fHBP into a single vaccine composition, therebyincreasing strain coverage, either as a mixture of separate proteins oras a fusion protein of the different variants (the latter being ‘tandemproteins’).

Reference 9 also reports a fHBP tandem protein (pages 18-19 of reference9). This tandem protein was purified and mixed with aluminium phosphateas an adjuvant, but it is reported not to adsorb well to the adjuvant.Good adsorption of the antigens is desirable, and it has been found thatsuch mixed fHBP proteins readily adsorb if aluminium hydroxide is usedas an adjuvant instead.

A problem when using aluminium hydroxide as an adjuvant, however, isthat it can degrade certain antigens. For instance, reference 10 reportsthat it can hydrolyse H. influenzae type B conjugate vaccines, even atlow temperatures, thus leading to reduced efficacy. Similarly,hydrolysis of S. typhi Vi capsular saccharide in the presence ofaluminium hydroxide is reported in reference 11. Thus it can bedesirable to formulate antigens using an adjuvant based on aluminiumphosphate, particularly if the adjuvanted vaccine may be mixed (eitherduring manufacture or at the time of use) with an antigen that may besusceptible to damage by an aluminium hydroxide e.g. a conjugatedbacterial capsular saccharide.

Thus there is a need to provide formulations of fHBP, and in particularof multiple fHBP variants, in which the fHBP(s) is/are adsorbed to anadjuvant but which do not require aluminium hydroxide.

DISCLOSURE OF THE INVENTION

The inventors have identified general techniques for achieving efficientadsorption of fHBP proteins to aluminium hydroxyphosphate adjuvants. Theuse of aluminium hydroxyphosphate can avoid the need to use aluminiumhydroxide, and the inventors' techniques avoid the inefficientadsorption described in reference 9. The adsorption techniques areparticularly useful for compositions which include multiple fHBPvariants.

In a first aspect of the invention, fHBP adsorption takes place at a pHwhich is equal to or below the aluminium hydroxyphosphate's point ofzero charge (PZC). For a given aluminium hydroxyphosphate adjuvant,therefore, an aqueous medium (e.g. buffer) would be selected with a pHequal to or below the adjuvant's PZC. Conversely, for a given pH analuminium hydroxyphosphate would be selected that has the same or ahigher PZC. This selection of pH and PZC can give immunogeniccompositions in which fHBP is stably adsorbed to an aluminiumhydroxyphosphate.

In a second aspect, a fHBP and an aluminium hydroxyphosphate adjuvantare selected such that the fHBP has an isoelectric point (pI) within therange of 5.0 to 7.0 (inclusive) and the adjuvant's PZC is selectedwithin the same range. By ensuring this close match of antigen andadjuvant characteristics it is possible to obtain stable adsorbedcompositions even if the adsorption pH is above the adjuvant's PZC.Stable adsorption is facilitated by the presence of a buffer which canmaintain pH also in the range of 5.0 to 7.0.

In a third aspect, if a fHBP has an isoelectric point above an aluminiumhydroxyphosphate adjuvant's PZC then a buffer is added to bring the pHto within 1.2 pH units of the PZC.

Thus, for the first aspect, the invention provides a method foradsorbing a meningococcal fHBP antigen to an aluminium hydroxyphosphateadjuvant, wherein adsorption takes place at a pH which is equal to orbelow the aluminium hydroxyphosphate's point of zero charge. Theadsorbed fHBP antigen can be used as an immunogen. The adsorption can beperformed in various ways. Mixing of fHBP antigen, aluminiumhydroxyphosphate and a buffer can occur in any suitable order, either bycombining all three components separately or by pre-mixing twocomponents and then mixing the pre-mix with the third component.

The invention also provides an immunogenic composition comprising ameningococcal fHBP antigen and an aluminium hydroxyphosphate adjuvant,wherein the aluminium hydroxyphosphate adjuvant has a point of zerocharge which is higher than the immunogenic composition's pH.

For the second aspect, the invention provides a method for adsorbing ameningococcal fHBP antigen to an aluminium hydroxyphosphate adjuvant,wherein (i) the meningococcal fHBP antigen has an isoelectric pointbetween 5.0 and 7.0, (ii) the aluminium hydroxyphosphate adjuvant has apoint of zero charge between 5.0 and 7.0, and (iii) adsorption of thefHBP antigens takes place at a pH between 5.0 and 7.0.

The invention also provides an immunogenic composition comprising ameningococcal fHBP antigen adsorbed to an aluminium hydroxyphosphateadjuvant, wherein (i) the meningococcal fHBP antigen has an isoelectricpoint between 5.0 and 7.0, and (ii) the aluminium hydroxyphosphateadjuvant has a point of zero charge between 5.0 and 7.0. The compositiontypically includes a buffer to maintain pH in the range of 5.0 to 7.0.

For the third aspect, the invention provides a method for adsorbing ameningococcal fHBP antigen to an aluminium hydroxyphosphate adjuvant,wherein (i) the meningococcal fHBP antigen has an isoelectric pointwhich is greater than the adjuvant's point of zero charge and (ii)adsorption takes place at a pH which is within 1.2 pH units of theadjuvant's point of zero charge. The pH during adsorption is preferablyachieved by including a buffer which maintains the pH within 1.2 pHunits of the adjuvant's point of zero charge.

The invention also provides an immunogenic composition comprising ameningococcal fHBP antigen adsorbed to an aluminium hydroxyphosphateadjuvant, wherein (i) the meningococcal fHBP antigen has an isoelectricpoint which is greater than the adjuvant's point of zero charge and (ii)the composition has a pH which is within 1.2 pH units of the adjuvant'spoint of zero charge. The composition may include a buffer whichmaintains the pH within 1.2 pH units of the adjuvant's PZC.

The invention is particularly useful in relation to compositions whichinclude more than one variant of fHBP. As mentioned above, suchcompositions have previously been reported not to adsorb well toaluminium adjuvants with phosphate groups.

Thus the invention provides a method for adsorbing two differentmeningococcal fHBP antigens to an aluminium hydroxyphosphate adjuvant,wherein adsorption of both of the fHBP antigens takes place at a pHwhich is equal to or below the aluminium hydroxyphosphate's point ofzero charge. The adsorbed fHBP antigens can be used for broad spectrummeningococcal immunisation. Mixing of fHBP antigens and aluminiumhydroxyphosphate (and a buffer) can occur in any suitable order.

The invention also provides an immunogenic composition comprising twodifferent meningococcal fHBP antigens, both of which are adsorbed toaluminium hydroxyphosphate adjuvant. The composition typically includesa buffer to control pH during and/or after adsorption.

The invention also provides an immunogenic composition comprising twodifferent meningococcal fHBP antigens and an aluminium hydroxyphosphateadjuvant, wherein the aluminium hydroxyphosphate adjuvant has a point ofzero charge which is higher than the immunogenic composition's pH.

The invention also provides a method for adsorbing two differentmeningococcal fHBP antigens to an aluminium hydroxyphosphate adjuvant,wherein (i) both of the meningococcal fHBP antigens have an isoelectricpoint between 5.0 and 7.0, (ii) the aluminium hydroxyphosphate adjuvanthas a point of zero charge between 5.0 and 7.0, and (iii) adsorption ofboth of the fHBP antigens takes place at a pH between 5.0 and 7.0.Adsorption may take place in the presence of a buffer.

The invention also provides an immunogenic composition comprising twodifferent meningococcal fHBP antigens, both of which are adsorbed toaluminium hydroxyphosphate adjuvant, wherein (i) both of themeningococcal fHBP antigens have an isoelectric point between 5.0 and7.0, (ii) the aluminium hydroxyphosphate adjuvant has a point of zerocharge between 5.0 and 7.0. The composition typically includes a bufferto maintain pH in the range of 5.0 to 7.0.

The invention also provides a method for adsorbing two differentmeningococcal fHBP antigens to an aluminium hydroxyphosphate adjuvant,wherein (i) the meningococcal fHBP antigens both have isoelectric pointswhich are greater than the adjuvant's point of zero charge and (ii)adsorption of each antigen takes place at a pH which is within 1.2 pHunits of the adjuvant's point of zero charge. The pH during adsorptionis preferably achieved by including a buffer which maintains the pHwithin 1.2 pH units of the adjuvant's point of zero charge.

The invention also provides an immunogenic composition comprising twodifferent meningococcal fHBP antigens, both adsorbed to aluminiumhydroxyphosphate adjuvant, wherein (i) each meningococcal fHBP antigenhas an isoelectric point which is greater than the adjuvant's point ofzero charge and (ii) the composition has a pH which is within 1.2 pHunits of the adjuvant's point of zero charge.

The invention also provides an immunogenic composition prepared by anyof the above methods.

In compositions of the invention, the or each fHBP antigen is preferablyat least 85% adsorbed, as described in more detail below.

Factor H Binding Protein(s)

Compositions of the invention include at least one meningococcal factorH binding protein (fHBP). Where a composition includes two differentfHBPs these are preferably different variants as disclosed in reference8. Different fHBPs will generate distinct immune responses which are notfully cross-reactive and which provide a broader spectrum of straincoverage against meningococci.

Where a composition comprises a single fHBP variant, it may include oneof the following:

-   -   (a) a first polypeptide comprising a first amino acid sequence,        where the first amino acid sequence comprises an amino acid        sequence (i) having at least a % sequence identity to SEQ ID NO:        1 and/or (ii) consisting of a fragment of at least x contiguous        amino acids from SEQ ID NO: 1;    -   (b) a second polypeptide, comprising a second amino acid        sequence, where the second amino acid sequence comprises an        amino acid sequence (i) having at least b % sequence identity to        SEQ ID NO: 2 and/or (ii) consisting of a fragment of at least y        contiguous amino acids from SEQ ID NO: 2;    -   (c) a third polypeptide, comprising a third amino acid sequence,        where the third amino acid sequence comprises an amino acid        sequence (i) having at least c % sequence identity to SEQ ID NO:        3 and/or (ii) consisting of a fragment of at least z contiguous        amino acids from SEQ ID NO: 3.

Where a composition comprises two different meningococcal fHBP antigens,it may include a combination of: (i) a first and second polypeptide asdefined above; (ii) a first and third polypeptide as defined above; or(iii) a second and third polypeptide as defined above. A combination ofa first and third polypeptide is preferred. A combination in which eachof the two different meningococcal fHBP antigens has a pI between 5.0and 7.0 is preferred, and in particular when they both have a pI in therange of 5.0 to 6.0 or in the range 5.2 to 6.2.

Where a composition comprises two different meningococcal fHBP antigens,although these may share some sequences in common, the first, second andthird polypeptides have different fHBP amino acid sequences.

A polypeptide comprising the first amino acid sequence will, whenadministered to a subject, elicit an antibody response comprisingantibodies that bind to the wild-type meningococcus protein havingnascent amino acid sequence SEQ ID NO: 20 (MC58). In some embodimentssome or all of these antibodies do not bind to the wild-typemeningococcus protein having nascent amino acid sequence SEQ ID NO: 21or to the wild-type meningococcus protein having nascent amino acidsequence SEQ ID NO: 22.

A polypeptide comprising the second amino acid sequence will, whenadministered to a subject, elicit an antibody response comprisingantibodies that bind to the wild-type meningococcus protein havingnascent amino acid sequence SEQ ID NO: 21 (2996). In some embodimentssome or all of these antibodies do not bind to the wild-typemeningococcus protein having nascent amino acid sequence SEQ ID NO: 20or to the wild-type meningococcus protein having nascent amino acidsequence SEQ ID NO: 22.

A polypeptide comprising the third amino acid sequence will, whenadministered to a subject, elicit an antibody response comprisingantibodies that bind to the wild-type meningococcus protein havingnascent amino acid sequence SEQ ID NO: 22 (M1239). In some embodimentssome or all of these antibodies do not bind to the wild-typemeningococcus protein having nascent amino acid sequence SEQ ID NO: 20or to the wild-type meningococcus protein having nascent amino acidsequence SEQ ID NO: 21.

In some embodiments the fragment of at least x contiguous amino acidsfrom SEQ ID NO: 1 is not also present within SEQ ID NO: 2 or within SEQID NO: 3. Similarly, the fragment of at least y contiguous amino acidsfrom SEQ ID NO: 2 might not also be present within SEQ ID NO: 1 orwithin SEQ ID NO: 3. Similarly, the fragment of at least z contiguousamino acids from SEQ ID NO: 3 might not also be present within SEQ IDNO: 1 or within SEQ ID NO: 2. In some embodiments, when said fragmentfrom one of SEQ ID NOs: 1 to 3 is aligned as a contiguous sequenceagainst the other two SEQ ID NOs, the identity between the fragment andeach of the other two SEQ ID NOs is less than 75% e.g. less than 70%,less than 65%, less than 60%, etc.

The value of a is at least 80 e.g. 82, 84, 86, 88, 90, 92, 94, 95, 96,97, 98, 99 or more. The value of b is at least 80 e.g. 82, 84, 86, 88,90, 92, 94, 95, 96, 97, 98, 99 or more. The value of c is at least 80e.g. 82, 84, 86, 88, 90, 92, 94, 95, 96, 97, 98, 99 or more. The valuesof a, b and c may be the same or different. In some embodiments, a b andc are identical.

The value of x is at least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60,70, 80, 90, 100, 120, 140, 160, 180, 200, 225, 250). The value of y isat least 7 e.g. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,120, 140, 160, 180, 200, 225, 250). The value of z is at least 7 e.g. 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 225, 250). The values of x, y and z may be the same or different.In some embodiments, x y and z are identical.

Fragments preferably comprise an epitope from the respective SEQ ID NO:sequence. Other useful fragments lack one or more amino acids (e.g. 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminusand/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25 or more) from the N-terminus of the respective SEQ ID NO: whileretaining at least one epitope thereof.

Amino acid sequences used with the invention may, compared to SEQ IDNOs: 1, 2 or 3, include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,etc.) conservative amino acid replacements i.e. replacements of oneamino acid with another which has a related side chain.Genetically-encoded amino acids are generally divided into fourfamilies: (1) acidic i.e. aspartate, glutamate; (2) basic i.e. lysine,arginine, histidine; (3) non-polar i.e. alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan; and (4)uncharged polar i.e. glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In general,substitution of single amino acids within these families does not have amajor effect on the biological activity. The polypeptides may have oneor more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) single amino aciddeletions relative to a reference sequence. The polypeptides may alsoinclude one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.)insertions (e.g. each of 1, 2, 3, 4 or 5 amino acids) relative to areference sequence.

A useful first amino acid sequence has at least 85% identity (e.g. >95%or 100%) to SEQ ID NO: 1. Another useful first amino acid sequence hasat least 95% identity (e.g. >98% or 100%) to SEQ ID NO: 4. Anotheruseful first amino acid sequence has at least 95% identity (e.g. >98% or100%) to SEQ ID NO: 5.

A useful third amino acid sequence has at least 85% identity (e.g. >95%or 100%) to SEQ ID NO: 3. Another useful third amino acid sequence hasat least 95% identity (e.g. >98% or 100%) to SEQ ID NO: 6.

Combinations comprising a mixture of first and third sequences basedaround SEQ ID NOs: 4 and 6 (or their close variants) are particularlyuseful. Another useful combination comprises a mixture of first andthird sequences based around a mixture of SEQ ID NOs: 5 and 6 (or theirclose variants). Thus a composition may comprise a polypeptidecomprising amino acid sequence SEQ ID NO: 23 and a polypeptidecomprising amino acid sequence SEQ ID NO: 25.

Where a composition includes two meningococcal fHBP antigens, this maybe in a bivalent fHBP composition, or there may be more than twodifferent fHBP antigens e.g. in a trivalent or tetravalent fHBPcomposition.

In some embodiments fHBP polypeptide(s) are lipidated e.g. at aN-terminus cysteine. In other embodiments, however, fHBP polypeptide(s)are not lipidated. For lipidated fHBPs, lipids attached to cysteineswill usually include palmitoyl residues e.g. astripalmitoyl-S-glyceryl-cysteine (Pam3Cys), dipalmitoyl-S-glycerylcysteine (Pam2Cys), N-acetyl (dipalmitoyl-S-glyceryl cysteine), etc.Examples of mature lipidated fHBP sequences are SEQ ID NO: 23 (includingSEQ ID NO: 4), SEQ ID NO: 24 (including SEQ ID NO: 5), and SEQ ID NO: 25(including SEQ ID NO: 6).

Administration of a fHBP will preferably elicit antibodies which canbind to a meningococcal polypeptide consisting of amino acid sequenceSEQ ID NO: 1, 2 or 3. Advantageous fHBP antigens for use with theinvention can elicit bactericidal anti-meningococcal antibodies afteradministration to a subject.

The total amount of a fHBP polypeptide will usually be between 1 and 500μg/dose e.g. between 60 and 200 μg/dose or between 120 and 500 μg/ml. Anamount of 20, 40, 50, 60, 80, 100 or 200 μg for each fHBP polypeptide istypical in a human vaccine dose. Thus a vaccine may be formulated toinclude this amount of each fHBP(s).

Where a composition comprises different meningococcal fHBP antigens,these may be present as separate polypeptides as described above (e.g. afirst and second polypeptide) or they may be present as part of a single‘hybrid’ polypeptide i.e. where at least two (e.g. 2, 3, 4, 5, or more)fHBP antigens are expressed as a single polypeptide chain, as disclosedfor meningococcal antigens in reference 12.

A hybrid polypeptide may comprise two or three of the following: a firstamino acid sequence as defined above; a second amino acid sequence asdefined above; and/or a third amino acid sequence as defined above.

Hybrid polypeptides can be represented by the formulaNH₂-A-{-X-L-}_(n)-B-COOH, wherein: X is a first, second or third aminoacid sequence as defined above; L is an optional linker amino acidsequence; A is an optional N-terminal amino acid sequence; B is anoptional C-terminal amino acid sequence; n is an integer of 2 or more(e.g. 2, 3, 4, 5, 6, etc.). Usually n is 2 or 3, and at least two of afirst, second and third amino acid sequence are present.

If a —X— moiety has a leader peptide sequence in its wild-type form,this may be included or omitted in the hybrid protein. In someembodiments, the leader peptides will be deleted except for that of the—X— moiety located at the N-terminus of the hybrid protein i.e. theleader peptide of X₁ will be retained, but the leader peptides of X₂ . .. X_(n), will be omitted. This is equivalent to deleting all leaderpeptides and using the leader peptide of X₁ as moiety -A-.

For each n instances of {—X-L-}, linker amino acid sequence -L- may bepresent or absent. For instance, when n=2 the hybrid may beNH₂—X₁-L₁-X₂-L₂-COOH, NH₂—X₁—X₂—COOH, NH₂—X₁-L₁-X₂—COOH,NH₂—X₁—X₂-L₂-COOH, etc. Linker amino acid sequence(s) -L- will typicallybe short (e.g. 20 or fewer amino acids i.e. 20, 19, 18, 17, 16, 15, 14,13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples comprise shortpeptide sequences which facilitate cloning, poly-glycine linkers (i.e.comprising Gly_(n) where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), andhistidine tags (i.e. His, where n=3, 4, 5, 6, 7, 8, 9, 10 or more).Other suitable linker amino acid sequences will be apparent to thoseskilled in the art. A useful linker is GSGGGG (SEQ ID NO: 15) orGSGSGGGG (SEQ ID NO:16), with the Gly-Ser dipeptide being formed from aBamHI restriction site, thus aiding cloning and manipulation, and the(Gly)₄ tetrapeptide being a typical poly-glycine linker. Anothersuitable linker, particularly for use as the final L_(n) is a Leu-Gludipeptide.

-A- is an optional N-terminal amino acid sequence. This will typicallybe short (e.g. 40 or fewer amino acids i.e. 40, 39, 38, 37, 36, 35, 34,33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples includeleader sequences to direct protein trafficking, or short peptidesequences which facilitate cloning or purification (e.g. histidine tagsi.e. His, where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitableN-terminal amino acid sequences will be apparent to those skilled in theart. If X₁ lacks its own N-terminus methionine, -A- is preferably anoligopeptide (e.g. with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) whichprovides a N-terminus methionine e.g. Met-Ala-Ser, or a single Metresidue.

-B- is an optional C-terminal amino acid sequence. This will typicallybe short (e.g. 40 or fewer amino acids i.e. 39, 38, 37, 36, 35, 34, 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples includesequences to direct protein trafficking, short peptide sequences whichfacilitate cloning or purification (e.g. comprising histidine tags i.e.His, where n=3, 4, 5, 6, 7, 8, 9, 10 or more, such as SEQ ID NO: 17), orsequences which enhance protein stability. Other suitable C-terminalamino acid sequences will be apparent to those skilled in the art.

Aluminium Hydroxyphosphate Adjuvants and Adsorption

Compositions of the invention include an aluminium hydroxyphosphateadjuvant. Such adjuvants are often referred to for convenience as“aluminium phosphate” [13], although hydroxyphosphates can bedistinguished from strict AlPO₄ by the presence of hydroxyl groups. Forexample, an IR spectrum band at 3164 cm⁻¹ (e.g. when heated to 200° C.)indicates the presence of structural hydroxyls. The aluminiumhydroxyphosphate adjuvant may contain a small amount of sulfate (i.e.aluminium hydroxyphosphate sulfate) and may also include sodium and/orchloride ions [14]. The adjuvant may be obtained by precipitation.

Aluminium hydroxyphosphate is not a stoichiometric compound and itshydroxyl and phosphate composition depends on precipitation reactantsand conditions. This hydroxyl/phosphate composition affects theadjuvant's point of zero charge (PZC; the pH at which a surface has zeronet charge). The PZC is inversely related to the degree of substitutionof phosphate for hydroxyl (the P/Al molar ratio). Substitution ofphosphate anions for hydroxyl anions lowers the PZC. Thus the PZC can bealtered by changing the concentration of free phosphate ions in solution(more phosphate=more acidic PZC) or by adding a buffer such as ahistidine buffer (makes PZC more basic). Aluminium hydroxyphosphatesused with the invention generally have a PZC of between 5.0 and 6.6 e.g.between 5.4 and 6.2.

The P/Al molar ratio of an aluminium hydroxyphosphate adjuvant willgenerally be between 0.3 and 1.2, preferably between 0.8 and 1.2, orbetween 0.85 and 1.0, and more preferably about 0.9. A P/Al molar ratioof at least 0.5 can provide an adjuvant with better aging properties.

The aluminium hydroxyphosphate will generally be amorphous (i.e.amorphous to X-rays). It will generally be particulate (e.g. plate-likemorphology as seen in transmission electron micrographs). Typicaldiameters of the plates are 10-100 nm, and these form aggregates sized0.5-20 μm (e.g. about 1-10 μm). Adsorptive capacities of between 0.7-1.5mg protein per mg Al⁺⁺⁺ at pH 7.4 have been reported for aluminiumhydroxyphosphate adjuvants.

A typical adjuvant is amorphous aluminium hydroxyphosphate with P/Almolar ratio between 0.84 and 0.92, and this adjuvant may be included at0.6 mg Al³⁺/ml.

The concentration of Al⁺⁺⁺ in a composition for administration to apatient is preferably less than 5 mg/ml e.g. ≦4 mg/ml, ≦3 mg/ml, ≦2mg/ml, ≦1 mg/ml, etc. A preferred range is between 0.2 and 1 mg/ml. Amaximum Al⁺⁺⁺ concentration of 0.85 mg/dose is preferred.

At least 85% (by weight) of a fHBP in a composition of the invention isadsorbed to aluminium hydroxyphosphate e.g. ≧90%, ≧95% or even 100%. Theproportion of adsorbed fHBP can be controlled by altering saltconcentration and/or pH during formulation e.g. in general, a higherNaCl concentration can decrease fHBP's adsorption. The amount ofadsorption for any formulation will depend on a combination ofparameters including the adjuvant's PZC, the salt concentration and pHduring formulation, the adjuvant concentration, the antigenconcentration and the antigen's pI. The impact of each of theseparameters on adsorption can be readily assessed. The degree ofadsorption can be determined by comparing the total amount of fHBPantigen in a composition (e.g. measured before adsorption occurs, ormeasured by desorbing adsorbed antigen) to the amount which remains inthe supernatant after centrifugation (e.g. see chapter 4 of ref. 15).The absence of detectable antigen in the supernatant aftercentrifugation indicates that total adsorption has occurred i.e. all ofthe fHBP is in the pellet, which contains the insoluble adjuvant and itsadsorbed content.

It is known to use mixtures of different aluminium salts in a singlevaccine e.g. see reference 16. Although adjuvants including bothaluminium hydroxyphosphate and hydroxide can be used with fHBP, it ispreferred that a composition should not include any aluminium hydroxideadjuvant because, as described above, it can degrade certain antigenswhich may be admixed with the fHBP (in particular, conjugated bacterialcapsular saccharides).

For the first aspect, the inventors have found that fHBP proteins can beefficiently adsorbed to an aluminium hydroxyphosphate adjuvant byensuring that adsorption takes place at a pH which is equal to or belowthe adjuvant's PZC. Thus an adjuvant can be chosen with a PZC equal toor above a desired formulation pH, or else a pH can be chosen equal toor below a desired adjuvant's PZC. Adjuvant and antigen are combinedunder these conditions and adsorption is allowed to occur. The pH shouldnot be so low as to prevent adsorption or to irreversibly denature thefHBP. Thus adsorption ideally occurs within 2 pH units (ideally within1.2 pH units) of the PZC.

For the second aspect, the inventors have found that fHBP proteins canbe efficiently adsorbed to an aluminium hydroxyphosphate adjuvant byusing a meningococcal fHBP antigen with an isoelectric point between 5.0and 7.0 and an aluminium hydroxyphosphate adjuvant with a point of zerocharge also between 5.0 and 7.0. Adsorption takes place at a pH between5.0 and 7.0, and pH may be maintained (before, during, and/or afteradsorption) by including a buffer to maintain pH in the range of 5.0 to7.0. Within the pH range of 5.0 and 7.0, a preferred sub-range is 5.0 to6.0. The second aspect is not suitable for all fHBPs as some (e.g. SEQID NO: 20) have a pI outside the required range, but an appropriate fHBPcan readily be selected.

The isoelectric point of a fHBP may be determined empirically by atechnique such as isoelectric focusing. More conveniently, however, theisoelectric point is a theoretical isoelectric point. This may becalculated using pKa values of amino acids described in reference 17e.g. using the relevant ExPASy tool [18]. For instance, nascent aminoacid sequence SEQ ID NO: 20 has a predicted pI of 7.72 whereas SEQ IDNOs: 21 and 22 have predicted pIs of 5.87 and 6.15. Mature sequences SEQID NOs: 23, 24 and 25 (comprising SEQ ID NOs: 4, 5 and 6, respectively)all have a predicted pI in the relevant range: 5.46, 5.72 and 5.86,respectively. A correction for a blocked N-terminus amine (e.g. whenlipidated) reduces the pI by about 0.1 but SEQ ID NOs: 23, 24 and 25still have predicted pIs in the range from 5.0 to 6.0. Combinationswhere each different meningococcal fHBP antigen has a pI between 5.0 and7.0 are preferred, and in particular when they both have a pI in therange of 5.0 to 6.0 or in the range 5.2 to 6.2.

A useful combination of fHBP antigens with pIs in the appropriate rangemay comprise a mixture of first and third sequences based around SEQ IDNOs: 4 and 6 (or their close variants) or a mixture of first and thirdsequences based around a mixture of SEQ ID NOs: 5 and 6 (or their closevariants). Further details of such antigen pairings are provided above.For example, a combination of SEQ ID NOs: 23 and 25 is particularlyuseful, and these two proteins may be lipidated (as discussed above).

For the third aspect, the inventors have found that a meningococcal fHBPantigen with a pI greater than an aluminium hydroxyphosphate adjuvant'sPZC can be efficiently adsorbed by ensuring that adsorption takes placeat a pH within 1.2 pH units of the PZC. Adsorption may take place at apH above or below the adjuvant's PZC, although the pH should not be soextreme as to irreversibly denature the fHBP. The pH during adsorptionis preferably achieved by including a buffer which maintains the pHwithin 1.2 pH units of the adjuvant's PZC. Where a pH is within 1.2 pHunits, it may be within 1 pH unit or less e.g. within 0.8 pH unit,within 0.6 pH unit, within or 0.5 pH unit.

Order of Mixing

As mentioned above, the invention provides a method for adsorbing ameningococcal fHBP antigen to an aluminium hydroxyphosphate adjuvant.Mixing of fHBP antigen(s), aluminium hydroxyphosphate and any buffer canoccur in any suitable order, either by combining all componentsseparately or by pre-mixing two components and then mixing the pre-mixwith the third component.

Thus, for example, in one embodiment the invention provides a processfor preparing an immunogenic composition comprising a meningococcal fHBPantigen, comprising a step of combining a meningococcal fHBP antigen andan aluminium hydroxyphosphate adjuvant, wherein: (i) the aluminiumhydroxyphosphate adjuvant has a point of zero charge; and (ii) thecombining step occurs at a pH lower than the point of zero charge suchthat the fHBP antigen adsorbs to the adjuvant.

In another embodiment, the invention provides a process for preparing animmunogenic composition comprising a meningococcal fHBP antigen,comprising a step of combining a meningococcal fHBP antigen and analuminium hydroxyphosphate adjuvant, wherein: (i) the aluminiumhydroxyphosphate adjuvant has a point of zero charge; and (ii) thecomposition has a pH lower than the point of zero charge, such that thefHBP antigen adsorbs to the adjuvant.

In another embodiment, the invention provides a process for preparing animmunogenic composition comprising a meningococcal fHBP antigen,comprising steps of: (i) providing an aqueous composition comprising ameningococcal fHBP antigen and having a pH; (ii) providing an aluminiumhydroxyphosphate adjuvant having a point of zero charge which is higherthan said pH; and (iii) combining the aqueous composition with thealuminium hydroxyphosphate adjuvant to give the immunogenic composition.

In another embodiment, the invention provides a process for preparing animmunogenic composition comprising a meningococcal fHBP antigen,comprising steps of: (i) providing an aqueous composition comprising analuminium hydroxyphosphate adjuvant and having a pH, wherein thealuminium hydroxyphosphate adjuvant has a point of zero charge which ishigher than said pH; and (ii) combining the aqueous composition with ameningococcal fHBP antigen to give the immunogenic composition.

In another embodiment, the invention provides a process for preparing animmunogenic composition comprising a meningococcal fHBP antigen,comprising steps of: (i) providing a first aqueous composition having apH; (ii) providing a second aqueous composition comprising ameningococcal fHBP antigen and an aluminium hydroxyphosphate adjuvanthaving a point of zero charge which is higher than said pH; and (iii)combining the first and second aqueous compositions to give theimmunogenic composition.

In another embodiment, the invention provides a process for preparing animmunogenic composition comprising a meningococcal fHBP antigen,comprising steps of: (i) providing a first aqueous composition having apH; (ii) providing a second aqueous composition comprising ameningococcal fHBP antigen; and (iii) providing an aluminiumhydroxyphosphate adjuvant having a point of zero charge which is higherthan said pH; and (iv) combining in any order the first aqueouscomposition, the second aqueous composition and the aluminiumhydroxyphosphate, to give the immunogenic composition.

The invention also provides a method for adsorbing two differentmeningococcal fHBP antigens to an aluminium hydroxyphosphate adjuvant,wherein adsorption of both of the fHBP antigens takes place at a pHwhich is equal to or below the aluminium hydroxyphosphate's point ofzero charge. Again, mixing of fHBP antigens, aluminium hydroxyphosphateand a buffer can occur in any suitable order.

Thus, in one embodiment, the two different fHBP antigens are separatelyadsorbed to aluminium hydroxyphosphate at the appropriate pH, and thetwo adsorbed antigens can then be mixed.

In another embodiment, the two different fHBP antigens are mixed witheach other and the mixture is then added to aluminium hydroxyphosphate,where the aluminium hydroxyphosphate is either at an appropriate pH foradsorption or where the pH is adjusted after addition of the mixture.

In another embodiment, the two different fHBP antigens are addedsequentially to aluminium hydroxyphosphate, where the aluminiumhydroxyphosphate is either at an appropriate pH for adsorption or wherethe pH is adjusted after addition of one or both fHBP antigens.

In another embodiment, one fHBP antigen is mixed with aluminiumhydroxyphosphate and then the other fHBP antigen is added to themixture, where the aluminium hydroxyphosphate is either at anappropriate pH for adsorption before addition of the first fHBP antigen,or where the pH is adjusted after addition of the first fHBP antigen, orwhere the pH is adjusted before addition of the second fHBP antigen, orwhere the pH is adjusted after addition of the second fHBP antigen.

These and other possibilities are available to the skilled person forall embodiments of the invention.

An Alternative Adjuvant

As an alternative to using an aluminium hydroxyphosphate adjuvant, theinvention can use a particulate complex of an immunostimulatoryoligonucleotide and a polycationic polymer, such as “IC31”. Definitionsgiven above can be amended accordingly. For instance, the inventionprovides an immunogenic composition comprising a meningococcal fHBPantigen and a particulate complex of an immunostimulatoryoligonucleotide and a polycationic polymer. The invention also providesan immunogenic composition comprising two different meningococcal fHBPantigens and a particulate complex of an immunostimulatoryoligonucleotide and a polycationic polymer.

Immunostimulatory oligonucleotides are known as useful adjuvants. Theyoften contain a CpG motif (a dinucleotide sequence containing anunmethylated cytosine linked to a guanosine) and their adjuvant effectis discussed in refs. 19-24. Oligonucleotides containing TpG motifs,palindromic sequences, multiple consecutive thymidine nucleotides (e.g.TTTT), multiple consecutive cytosine nucleotides (e.g. CCCC) or poly(dG)sequences are also known immunostimulants, as are double-stranded RNAs.Although any of these various immunostimulatory oligonucleotides can beused with the invention, it is preferred to use an oligodeoxynucleotidecontaining deoxyinosine and/or deoxyuridine, and ideally anoligodeoxynucleotide containing deoxyinosine and deoxycytosine.Inosine-containing oligodeoxynucleotides may include a CpI motif (adinucleotide sequence containing a cytosine linked to an inosine). Theoligodeoxynucleotide may include more than one (e.g. 2, 3, 4, 5, 6 ormore) CpI motif, and these may be directly repeated (e.g. comprising thesequence (CI)_(x), where x is 2, 3, 4, 5, 6 or more) or separated fromeach other (e.g. comprising the sequence (CIN)_(x), where x is 2, 3, 4,5, 6 or more, and where each N independently represents one or morenucleotides). Cytosine residues are ideally unmethylated.

An oligonucleotide will typically have between 10 and 100 nucleotidese.g. 15-50 nucleotides, 20-30 nucleotides, or 25-28 nucleotides. It willtypically be single-stranded.

The oligonucleotide can include exclusively natural nucleotides,exclusively non-natural nucleotides, or a mix of both. For instance, itmay include one or more phosphorothioate linkage(s), and/or one or morenucleotides may have a 2′-O-methyl modification.

A preferred oligonucleotide is a single-stranded deoxynucleotidecomprising the 26-mer sequence 5′-(IC)₁₃-3′ (SEQ ID NO: 18). Thisoligodeoxynucleotide forms stable complexes with polycationic polymersto give a good adjuvant.

The polycationic polymer is ideally a polycationic peptide. The polymermay include one or more leucine amino acid residue(s) and/or one or morelysine amino acid residue(s). The polymer may include one or morearginine amino acid residue(s). It may include at least one directrepeat of one of these amino acids e.g. one or more Leu-Leu dipeptidesequence(s), one or more Lys-Lys dipeptide sequence(s), or one or moreArg-Arg dipeptide sequence(s). It may include at least one (andpreferably multiple e.g. 2 or 3) Lys-Leu dipeptide sequence(s) and/or atleast one (and preferably multiple e.g. 2 or 3) Lys-Leu-Lys tripeptidesequence(s).

The peptide may comprise a sequence R₁—XZXZ_(x)XZX—R₂, wherein: x is 3,4, 5, 6 or 7; each X is independently a positively-charged naturaland/or non-natural amino acid residue; each Z is independently an aminoacid residue L, V, I, F or W; and R₁ and R₂ are independently selectedfrom the group consisting of —H, —NH₂, —COCH₃, or —COH. In someembodiments X—R₂ may be an amide, ester or thioester of the peptide'sC-terminal amino acid residue.

A polycationic peptide will typically have between 5 and 50 amino acidse.g. 6-20 amino acids, 7-15 amino acids, or 9-12 amino acids.

A peptide can include exclusively natural amino acids, exclusivelynon-natural amino acids, or a mix of both. It may include L-amino acidsand/or D-amino acids. L-amino acids are typical.

A peptide can have a natural N-terminus (NH₂—) or a modified N-terminuse.g. a hydroxyl, acetyl, etc. A peptide can have a natural C-terminus(—COOH) or a modified C-terminus e.g. a hydroxyl, an acetyl, etc. Suchmodifications can improve the peptide's stability.

A preferred peptide for use with the invention is the 11-mer KLKLLLLLKLK(SEQ ID NO: 19), with all L-amino acids. The N-terminus may bedeaminated and the C-terminus may be hydroxylated. A preferred peptideis H-KLKL₅KLK-OH, with all L-amino acids. This oligopeptide forms stablecomplexes with immunostimulatory oligonucleotides to give a goodadjuvant.

The most preferred mixture of immunostimulatory oligonucleotide andpolycationic polymer is the TLR9 agonist known as IC31™ [25-27], whichis an adsorptive complex of oligodeoxynucleotide SEQ ID NO: 18 andpolycationic oligopeptide SEQ ID NO: 19.

The oligonucleotide and oligopeptide can be mixed together at variousratios, but they will generally be mixed with the peptide at a molarexcess. The molar excess may be at least 5:1 e.g. 10:1, 15:1, 20:1,25:1, 30;1, 35:1, 40:1 etc. A molar ratio of about 25:1 is ideal[28,29]. Mixing at this excess ratio can result in formation ofinsoluble particulate complexes between oligonucleotide andoligopeptide. The complexes can be combined with an oil-in-wateremulsion.

The oligonucleotide and oligopeptide will typically be mixed underaqueous conditions e.g. a solution of the oligonucleotide can be mixedwith a solution of the oligopeptide with a desired ratio. The twosolutions may be prepared by dissolving dried (e.g. lyophilised)materials in water or buffer to form stock solutions that can then bemixed. The complexes can be analysed using the methods disclosed inreference 30.

Poly-arginine and CpG oligodeoxynucleotides similarly form complexes[31] which may be used.

The complexes can be maintained in aqueous suspension e.g. in water orin buffer. Typical buffers for use with the complexes are phosphatebuffers (e.g. phosphate-buffered saline), Tris buffers, Tris/sorbitolbuffers, borate buffers, succinate buffers, citrate buffers, histidinebuffers, etc. As an alternative, complexes may sometimes be lyophilised.

Various concentrations of oligonucleotide and polycationic polymer canbe used e.g. any of the concentrations used in references 25, 28 or 29.For example, a polycationic oligopeptide can be present at 1100 μM, 1000μM, 350 μM, 220 μM, 200 μM, 110 μM, 100 μM, 11 μM, 10 μM, etc. Anoligonucleotide can be present at 44 nM, 40 nM, 14 nM, 4.4 nM, 4 nM,etc. A polycationic oligopeptide concentration of less than 2000 nM istypical. For SEQ ID NOs: 18 & 19, mixed at a molar ratio of 1:25, theconcentrations in mg/mL in three embodiments of the invention may thusbe 0.311 & 1.322, or 0.109 & 0.463, or 0.031 and 0.132.

In embodiments of the invention which include a particulate complex ofan immunostimulatory oligonucleotide and a polycationic polymer, it isuseful if this complex is the sole adjuvant e.g. the composition may befree from aluminium salts and free from oil-in-water emulsions.

In a specific embodiment, the invention provides an immunogeniccomposition comprising: a particulate complex of an immunostimulatoryoligonucleotide and a polycationic polymer (e.g. IC31); a meningococcalfHBP antigen; and conjugated capsular saccharides from 1, 2, 3 or 4 ofmeningococcal serogroups A, C, W135 and/or Y. Further details ofsuitable conjugated saccharides are given below.

Further Antigen(s)

In addition to fHBP antigen(s), compositions of the invention caninclude further antigens from meningococcus or from other pathogens e.g.from other bacteria such as pneumococcus.

Further Meningococcal Polypeptide Antigens

In addition to including meningococcal fHBP polypeptide antigen(s), acomposition may include one or more further meningococcal polypeptideantigen(s). Thus a composition may include a polypeptide antigenselected from the group consisting of: 287, NadA, NspA, HmbR, NhhA, App,and/or Omp85. These antigens will usefully be present as purifiedpolypeptides e.g. recombinant polypeptides. The antigen will preferablyelicit bactericidal anti-meningococcal antibodies after administrationto a subject. If a composition includes a PorA antigen then, in someembodiments, only one meningococcal PorA serosubtype is included. Insome embodiments, no meningococcal PorA outer membrane protein isincluded in a composition.

A composition of the invention may include a 287 antigen. The 287antigen was included in the published genome sequence for meningococcalserogroup B strain MC58 [32] as gene NMB2132 (GenBank accession numberGI:7227388; SEQ ID NO: 9 herein). The sequences of 287 antigen from manystrains have been published since then. For example, allelic forms of287 can be seen in FIGS. 5 and 15 of reference 33, and in example 13 andFIG. 21 of reference 34 (SEQ IDs 3179 to 3184 therein). Variousimmunogenic fragments of the 287 antigen have also been reported.Preferred 287 antigens for use with the invention comprise an amino acidsequence: (a) having 50% or more identity (e.g. 60%, 650%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% ormore) to SEQ ID NO: 9; and/or (b) comprising a fragment of at least ‘n’consecutive amino acids of SEQ ID NO: 9, wherein ‘n’ is 7 or more (e.g.8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 250 or more). Preferred fragments of (b) comprise an epitope fromSEQ ID NO: 9. The most useful 287 antigens of the invention can elicitantibodies which, after administration to a subject, can bind to ameningococcal polypeptide consisting of amino acid sequence SEQ ID NO:9. Advantageous 287 antigens for use with the invention can elicitbactericidal anti-meningococcal antibodies after administration to asubject.

A composition of the invention may include a NadA antigen. The NadAantigen was included in the published genome sequence for meningococcalserogroup B strain MC58 [32] as gene NMB1994 (GenBank accession numberGI:7227256; SEQ ID NO: 10 herein). The sequences of NadA antigen frommany strains have been published since then, and the protein's activityas a Neisserial adhesin has been well documented. Various immunogenicfragments of NadA have also been reported. Preferred NadA antigens foruse with the invention comprise an amino acid sequence: (a) having 50%or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 10; and/or(b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQID NO: 10, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25,30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferredfragments of (b) comprise an epitope from SEQ ID NO: 10. The most usefulNadA antigens of the invention can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 10. Advantageous NadAantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject. SEQ IDNO: 6 is one such fragment.

A composition of the invention may include a NspA antigen. The NspAantigen was included in the published genome sequence for meningococcalserogroup B strain MC58 [32] as gene NMB0663 (GenBank accession numberGI:7225888; SEQ ID NO: 11 herein). The antigen was previously known fromreferences 35 & 36. The sequences of NspA antigen from many strains havebeen published since then. Various immunogenic fragments of NspA havealso been reported. Preferred NspA antigens for use with the inventioncomprise an amino acid sequence: (a) having 50% or more identity (e.g.60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5% or more) to SEQ ID NO: 11; and/or (b) comprising afragment of at least ‘n’ consecutive amino acids of SEQ ID NO: 11,wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferred fragmentsof (b) comprise an epitope from SEQ ID NO: 11. The most useful NspAantigens of the invention can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 11. Advantageous NspAantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

Compositions of the invention may include a meningococcal HmbR antigen.The full-length HmbR sequence was included in the published genomesequence for meningococcal serogroup B strain MC58 [32] as gene NMB1668(SEQ ID NO: 7 herein). Reference 37 reports a HmbR sequence from adifferent strain (SEQ ID NO: 8 herein). SEQ ID NOs: 7 and 8 differ inlength by 1 amino acid and have 94.2% identity. The invention can use apolypeptide that comprises a full-length HmbR sequence, but it willoften use a polypeptide that comprises a partial HmbR sequence. Thus insome embodiments a HmbR sequence used according to the invention maycomprise an amino acid sequence having at least i % sequence identity toSEQ ID NO: 7, where the value of i is 50, 60, 70, 80, 90, 95, 99 ormore. In other embodiments a HmbR sequence used according to theinvention may comprise a fragment of at least j consecutive amino acidsfrom SEQ ID NO: 7, where the value of j is 7, 8, 10, 12, 14, 16, 18, 20,25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more. In otherembodiments a HmbR sequence used according to the invention may comprisean amino acid sequence (i) having at least i % sequence identity to SEQID NO: 7 and/or (ii) comprising a fragment of at least j consecutiveamino acids from SEQ ID NO: 7. Preferred fragments of j amino acidscomprise an epitope from SEQ ID NO: 7. Such epitopes will usuallycomprise amino acids that are located on the surface of HmbR. Usefulepitopes include those with amino acids involved in HmbR's binding tohaemoglobin, as antibodies that bind to these epitopes can block theability of a bacterium to bind to host haemoglobin. The topology ofHmbR, and its critical functional, residues, were investigated inreference 38. The most useful HmbR antigens of the invention can elicitantibodies which, after administration to a subject, can bind to ameningococcal polypeptide consisting of amino acid sequence SEQ ID NO:7. Advantageous HmbR antigens for use with the invention can elicitbactericidal anti-meningococcal antibodies after administration to asubject.

A composition of the invention may include a NhhA antigen. The NhhAantigen was included in the published genome sequence for meningococcalserogroup B strain MC58 [32] as gene NMB0992 (GenBank accession numberGI:7226232; SEQ ID NO: 12 herein). The sequences of NhhA antigen frommany strains have been published since e.g. refs 33 & 39, and variousimmunogenic fragments of NhhA have been reported. It is also known asHsf. Preferred NhhA antigens for use with the invention comprise anamino acid sequence: (a) having 50% or more identity (e.g. 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5% or more) to SEQ ID NO: 12; and/or (b) comprising a fragment of atleast ‘n’ consecutive amino acids of SEQ ID NO: 12, wherein ‘n’ is 7 ormore (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80,90, 100, 150, 200, 250 or more). Preferred fragments of (b) comprise anepitope from SEQ ID NO: 12. The most useful NhhA antigens of theinvention can elicit antibodies which, after administration to asubject, can bind to a meningococcal polypeptide consisting of aminoacid sequence SEQ ID NO: 12. Advantageous NhhA antigens for use with theinvention can elicit bactericidal anti-meningococcal antibodies afteradministration to a subject.

A composition of the invention may include an App antigen. The Appantigen was included in the published genome sequence for meningococcalserogroup B strain MC58 [32] as gene NMB1985 (GenBank accession numberGI:7227246; SEQ ID NO: 13 herein). The sequences of App antigen frommany strains have been published since then. Various immunogenicfragments of App have also been reported. Preferred App antigens for usewith the invention comprise an amino acid sequence: (a) having 50% ormore identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 13; and/or(b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQID NO: 13, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25,30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferredfragments of (b) comprise an epitope from SEQ ID NO: 13. The most usefulApp antigens of the invention can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 13. Advantageous Appantigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

A composition of the invention may include an Omp85 antigen. The Omp85antigen was included in the published genome sequence for meningococcalserogroup B strain MC58 [32] as gene NMB0182 (GenBank accession numberGI:7225401; SEQ ID NO: 14 herein). The sequences of Omp85 antigen frommany strains have been published since then. Further information onOmp85 can be found in references 40 and 41. Various immunogenicfragments of Omp85 have also been reported. Preferred Omp85 antigens foruse with the invention comprise an amino acid sequence: (a) having 50%or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 14; and/or(b) comprising a fragment of at least ‘n’ consecutive amino acids of SEQID NO: 14, wherein ‘n’ is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25,30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or more). Preferredfragments of (b) comprise an epitope from SEQ ID NO: 14. The most usefulOmp85 antigens of the invention can elicit antibodies which, afteradministration to a subject, can bind to a meningococcal polypeptideconsisting of amino acid sequence SEQ ID NO: 14. Advantageous Omp85antigens for use with the invention can elicit bactericidalanti-meningococcal antibodies after administration to a subject.

Meningococcal Lipooligosaccharide

In addition to including meningococcal fHBP polypeptide antigen(s), acomposition may include one or more meningococcal lipooligosaccharide(LOS) antigen(s). Meningococcal LOS is a glucosamine-based phospholipidthat is found in the outer monolayer of the outer membrane of thebacterium. It includes a lipid A portion and a core oligosaccharideregion, with the lipid A portion acting as a hydrophobic anchor in themembrane. Heterogeneity within the oligosaccharide core generatesstructural and antigenic diversity among different meningococcalstrains, which has been used to subdivide the strains into 12immunotypes (L1 to L12). The invention may use LOS from any immunotypee.g. from L1, L2, L3, L4, L5, L6, L7 and/or L8.

The L2 and L3 α-chains naturally include lacto-N-neotetraose (LNnT).Where the invention uses LOS from a L2 or L3 immunotype this LNnT may beabsent. This absence can be achieved conveniently by using mutantstrains that are engineered to disrupt their ability to synthesise theLNnT tetrasaccharide within the α-chain. It is known to achieve thisgoal by knockout of the enzymes that are responsible for the relevantbiosynthetic additions [42,43]. For instance, knockout of the LgtBenzyme prevents addition of the terminal galactose of LNnT, as well aspreventing downstream addition of the α-chain's terminal sialic acid.Knockout of the LgtA enzyme prevents addition of theN-acetyl-glucosamine of LNnT, and also the downstream additions. LgtAknockout may be accompanied by LgtC knockout. Similarly, knockout of theLgtE and/or GalE enzyme prevents addition of internal galactose, andknockout of LgtF prevents addition of glucose to the Hep¹ residue. Anyof these knockouts can be used, singly or in combination, to disrupt theLNnT tetrasaccharide in a L2, L3, L4, L7 or L9 immunotype strain.Knockout of at least LgtB is preferred, as this provides a LOS thatretains useful immunogenicity while removing the LNnT epitope.

In addition to, or in place of, mutations to disrupt the LNnT epitope, aknockout of the galE gene also provides a useful modified LOS, and alipid A fatty transferase gene may similarly be knocked out [44]. Atleast one primary O-linked fatty acid may be removed from LOS [45]. LOShaving a reduced number of secondary acyl chains per LOS molecule canalso be used [46]. The LOS will typically include at least theGlcNAc-Hep₂phosphoethanolamine-KDO₂-Lipid A structure [47]. The LOS mayinclude a GlcNAcβ1-3Galβ1-4Glc trisaccharide while lacking the LNnTtetrasaccharide.

LOS may be included in compositions of the invention in various forms.It may be used in purified form on its own. It may be conjugated to acarrier protein. When LOS is conjugated, conjugation may be via a lipidA portion in the LOS or by any other suitable moiety e.g. its KDOresidues. If the lipid A moiety of LOS is absent then such alternativelinking is required. Conjugation techniques for LOS are known from e.g.references 45, 47, 48, 49, etc. Useful carrier proteins for theseconjugates are discussed below e.g. bacterial toxins, such as diphtheriaor tetanus toxins, or toxoids or mutants thereof.

The LOS may be from a strain (e.g. a genetically-engineeredmeningococcal strain) which has a fixed (i.e. not phase variable) LOSimmunotype as described in reference 50. For example, L2 and L3 LOSimmunotypes may be fixed. Such strains may have a rate of switchingbetween immunotypes that is reduced by more than 2-fold (even >50_fold)relative to the original wild-type strain. Reference 50 discloses howthis result can be achieved by modification of the lgtA and/or lgtG geneproducts.

LOS may be O-acetylated on a GlcNac residue attached to its Heptose IIresidue e.g. for L3 [51].

An immunogenic composition can include more than one type of LOS e.g.LOS from meningococcal immunotypes L2 and L3. For example, the LOScombinations disclosed in reference 52 may be used.

A LOS antigen can preferably elicit bactericidal anti-meningococcalantibodies after administration to a subject.

However, preferred compositions of the invention are free frommeningococcal lipooligosaccharide.

Meningococcal Capsular Saccharide Antigen(s)

In addition to including meningococcal fHBP polypeptide antigen(s), acomposition may include one or more meningococcal capsular saccharideconjugates. A composition of the invention may include one or moreconjugates of capsular saccharides from 1, 2, 3, or 4 of meningococcalserogroups A, C, W135 and Y e.g. A+C, A+W135, A+Y, C+W135, C+Y, W135+Y,A+C+W135, A+C+Y, A+W135+Y, A+C+W135+Y, etc. Compositions including aconjugated serogroup C capsular saccharide are useful, and compositionsincluding saccharides from all four of serogroups A, C, W135 and Y areideal.

The capsular saccharide of serogroup A meningococcus is a homopolymer of(α1→6)-linked N-acetyl-D-mannosamine-1-phosphate, with partialO-acetylation in the C3 and C4 positions. Acetylation at the C-3position can be 70-95%. Conditions used to purify the saccharide canresult in de-O-acetylation (e.g. under basic conditions), but it isuseful to retain OAc at this C-3 position. In some embodiments, at least50% (e.g. at least 60%, 70%, 80%, 90%, 95% or more) of the mannosamineresidues in a serogroup A saccharides are O-acetylated at the C-3position. Acetyl groups can be replaced with blocking groups to preventhydrolysis [53], and such modified saccharides are still serogroup Asaccharides within the meaning of the invention.

The serogroup C capsular saccharide is a homopolymer of (α2→9)-linkedsialic acid (N-acetyl neuraminic acid, or ‘NeuNAc’). The saccharidestructure is written as →9)-Neu p NAc 7/8 OAc− (α2→. Most serogroup Cstrains have O-acetyl groups at C-7 and/or C-8 of the sialic acidresidues, but about 15% of clinical isolates lack these O-acetyl groups[54,55]. The presence or absence of OAc groups generates uniqueepitopes, and the specificity of antibody binding to the saccharide mayaffect its bactericidal activity against O-acetylated (OAc+) andde-O-acetylated (OAc−) strains [56-58]. Serogroup C saccharides usedwith the invention may be prepared from either OAc+ or OAc− strains.Licensed MenC conjugate vaccines include both OAc− (NEISVAC-C™) and OAc+(MENJUGATE™ & MENINGITEC™) saccharides. In some embodiments, strains forproduction of serogroup C conjugates are OAc+ strains, e.g. of serotype16, serosubtype P1.7a,1, etc. Thus C:16:P1.7a,1 OAc+ strains may beused. OAc+ strains in serosubtype P1.1 are also useful, such as the C11strain.

The serogroup W135 saccharide is a polymer of sialic acid-galactosedisaccharide units. Like the serogroup C saccharide, it has variableO-acetylation, but at sialic acid 7 and 9 positions [59]. The structureis written as: →4)-D-Neup5Ac(7/9OAc)-α-(2→6)-D-Gal-α-(1→.

The serogroup Y saccharide is similar to the serogroup W135 saccharide,except that the disaccharide repeating unit includes glucose instead ofgalactose. Like serogroup W135, it has variable O-acetylation at sialicacid 7 and 9 positions [59]. The serogroup Y structure is written as:→4)-D-Neup5Ac(7/9OAc)-α-(2→6)-D-Glc-α-(1→.

The saccharides used according to the invention may be O-acetylated asdescribed above (e.g. with the same O-acetylation pattern as seen innative capsular saccharides), or they may be partially or totallyde-O-acetylated at one or more positions of the saccharide rings, orthey may be hyper-O-acetylated relative to the native capsularsaccharides.

The saccharide moieties in conjugates may comprise full-lengthsaccharides as prepared from meningococci, and/or may comprise fragmentsof full-length saccharides i.e. the saccharides may be shorter than thenative capsular saccharides seen in bacteria. The saccharides may thusbe depolymerised, with depolymerisation occurring during or aftersaccharide purification but before conjugation. Depolymerisation reducesthe chain length of the saccharides. One depolymerisation methodinvolves the use of hydrogen peroxide. Hydrogen peroxide is added to asaccharide (e.g. to give a final H₂O₂ concentration of 1%), and themixture is then incubated (e.g. at about 55° C.) until a desired chainlength reduction has been achieved. Another depolymerisation methodinvolves acid hydrolysis. Other depolymerisation methods are known inthe art. The saccharides used to prepare conjugates for use according tothe invention may be obtainable by any of these depolymerisationmethods. Depolymerisation can be used in order to provide an optimumchain length for immunogenicity and/or to reduce chain length forphysical manageability of the saccharides. In some embodiments,saccharides have the following range of average degrees ofpolymerisation (Dp): A=10-20; C=12-22; W135=15-25; Y=15-25. In terms ofmolecular weight, rather than Dp, useful ranges are, for all serogroups:<100 kDa; 5 kDa-75 kDa; 7 kDa-50 kDa; 8 kDa-35 kDa; 12 kDa-25 kDa; 15kDa-22 kDa.

In some embodiments, the average molecular weight for saccharides fromeach of meningococcal serogroups A, C, W135 and Y may be more than 50kDa e.g. ≧75 kDa, ≧100 kDa, ≧110 kDa, ≧120 kDa, ≧130 kDa, etc. [60], andeven up to 1500 kDa, in particular as determined by MALLS. For instance:a MenA saccharide may be in the range 50-500 kDa e.g. 60-80 kDa; a MenCsaccharide may be in the range 100-210 kDa; a MenW135 saccharide may bein the range 60-190 kDa e.g. 120-140 kDa; and/or a MenY saccharide maybe in the range 60-190 kDa e.g. 150-160 kDa.

The mass of meningococcal saccharide per serogroup in a composition willusually be between 1 μg and 20 μg e.g. between 2 and 10 μg perserogroup, or about 4 μg or about 5 μg or about 10 μg. Where conjugatesfrom more than one serogroup are included then they may be present atsubstantially equal masses e.g. the mass of each serogroup's saccharideis within +10% of each other. As an alternative to an equal ratio, adouble mass of serogroup A saccharide may be used. Thus a vaccine mayinclude MenA saccharide at 10 μg and MenC, W135 and Y saccharides at 5μg each.

Useful carrier proteins for meningococcal conjugates include bacterialtoxins, such as diphtheria or tetanus toxins, or toxoids or mutantsthereof. These are commonly used in conjugate vaccines. For example, theCRM197 diphtheria toxin mutant is useful [61]. Other suitable carrierproteins include synthetic peptides [62,63], heat shock proteins[64,65], pertussis proteins [66,67], cytokines [68], lymphokines [68],hormones [68], growth factors [68], artificial proteins comprisingmultiple human CD4⁺ T cell epitopes from various pathogen-derivedantigens [69] such as N19 [70], protein D from H. influenzae [71-73],pneumolysin [74] or its non-toxic derivatives [75], pneumococcal surfaceprotein PspA [76], iron-uptake proteins [77], toxin A or B from C.difficile [78], recombinant Pseudomonas aeruginosa exoprotein A (rEPA)[79], etc. CRM197 is preferred.

Where a composition includes conjugates from more than one meningococcalserogroup it is possible to use the same carrier protein for eachseparate conjugate, or to use different carrier proteins. In both cases,though, a mixture of different conjugates will usually be formed bypreparing each serotype conjugate separately, and then mixing them toform a mixture of separate conjugates.

Conjugates with a saccharide:protein ratio (w/w) of between 1:5 (i.e.excess protein) and 5:1 (i.e. excess saccharide) may be used e.g. ratiosbetween 1:2 and 5:1 and ratios between 1:1.25 and 1:2.5. As described inreference 80, different meningococcal serogroup conjugates in a mixturecan have different saccharide:protein ratios e.g. one may have a ratioof between 1:2 & 1:5, whereas another has a ratio between 5:1 & 1:1.99.

A carrier protein may be covalently conjugated to a meningococcalsaccharide directly or via a linker. Various linkers are known. Forexample, attachment may be via a carbonyl, which may be formed byreaction of a free hydroxyl group of a modified saccharide with CDI[81,82] followed by reaction with a protein to form a carbamate linkage.Carbodiimide condensation can be used [83]. An adipic acid linker can beused, which may be formed by coupling a free —NH₂ group (e.g. introducedto a saccharide by amination) with adipic acid (using, for example,diimide activation), and then coupling a protein to the resultingsaccharide-adipic acid intermediate [84,85]. Other linkers includeβ-propionamido [86], nitrophenyl-ethylamine [87], haloacyl halides [88],glycosidic linkages [89], 6-aminocaproic acid [90],N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) [91], adipic aciddihydrazide ADH [92], C₄ to C₁₂ moieties [93], etc.

Conjugation via reductive amination can be used. The saccharide mayfirst be oxidised with periodate to introduce an aldehyde group, whichcan then form a direct covalent linkage to a carrier protein viareductive amination e.g. to the ε-amino group of a lysine. If thesaccharide includes multiple aldehyde groups per molecule then thislinkage technique can lead to a cross-linked product, where multiplealdehydes react with multiple carrier amines.

As described in reference 94, a mixture can include one conjugate withdirect saccharide/protein linkage and another conjugate with linkage viaa linker. This arrangement applies particularly when using saccharideconjugates from different meningococcal serogroups e.g. MenA and MenCsaccharides may be conjugated via a linker, whereas MenW135 and MenYsaccharides may be conjugated directly to a carrier protein.

A meningococcal saccharide may comprise a full-length intact saccharideas prepared from meningococcus, and/or may comprise fragments offull-length saccharides i.e. the saccharides may be shorter than thenative capsular saccharides seen in bacteria. The saccharides may thusbe depolymerised, with depolymerisation occurring during or aftersaccharide purification but before conjugation. Depolymerisation reducesthe chain length of the saccharides. Depolymerisation can be used inorder to provide an optimum chain length for immunogenicity and/or toreduce chain length for physical manageability of the saccharides.

Conjugated Pneumococcal Capsular Saccharide(s)

Compositions of the invention may include a pneumococcal capsularsaccharide conjugated to a carrier protein.

The invention can include capsular saccharide from one or more differentpneumococcal serotypes. Where a composition includes saccharide antigensfrom more than one serotype, these are preferably prepared separately,conjugated separately, and then combined. Methods for purifyingpneumococcal capsular saccharides are known in the art (e.g. seereference 95) and vaccines based on purified saccharides from 23different serotypes have been known for many years. Improvements tothese methods have also been described e.g. for serotype 3 as describedin reference 96, or for serotypes 1, 4, 5, 6A, 6B, 7F and 19A asdescribed in reference 97.

Pneumococcal capsular saccharide(s) will typically be selected from thefollowing serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A,12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and/or 33F. Thus, intotal, a composition may include a capsular saccharide from 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 ormore different serotypes.

A useful combination of serotypes is a 7-valent combination e.g.including capsular saccharide from each of serotypes 4, 6B, 9V, 14, 18C,19F, and 23F. Another useful combination is a 9-valent combination e.g.including capsular saccharide from each of serotypes 1, 4, 5, 6B, 9V,14, 18C, 19F and 23F. Another useful combination is a 10-valentcombination e.g. including capsular saccharide from each of serotypes 1,4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. An 11-valent combination mayfurther include saccharide from serotype 3. A 12-valent combination mayadd to the 10-valent mixture: serotypes 6A and 19A; 6A and 22F; 19A and22F; 6A and 15B; 19A and 15B; or 22F and 15B. A 13-valent combinationmay add to the 11-valent mixture: serotypes 19A and 22F; 8 and 12F; 8and 15B; 8 and 19A; 8 and 22F; 12F and 15B; 12F and 19A; 12F and 22F;15B and 19A; 15B and 22F; 6A and 19A, etc.

Thus a useful 13-valent combination includes capsular saccharide fromserotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19, 19F and 23F e.g.prepared as disclosed in references 98 to 101. One such combinationincludes serotype 6B saccharide at about 8 μg/ml and the other 12saccharides at concentrations of about 4 μg/ml each. Another suchcombination includes serotype 6A and 6B saccharides at about 8 μg/mleach and the other 11 saccharides at about 4 μg/ml each.

Suitable carrier proteins for conjugates are discussed above in relationto meningococcal conjugates. Particularly useful carrier proteins forpneumococcal conjugate vaccines are CRM197, tetanus toxoid, diphtheriatoxoid and H. influenzae protein D. CRM197 is used in PREVNAR™. A13-valent mixture may use CRM197 as the carrier protein for each of the13 conjugates, and CRM197 may be present at about 55-60 μg/ml.

Where a composition includes conjugates from more than one pneumococcalserotype, it is possible to use the same carrier protein for eachseparate conjugate, or to use different carrier proteins. In both cases,though, a mixture of different conjugates will usually be formed bypreparing each serotype conjugate separately, and then mixing them toform a mixture of separate conjugates. Reference 102 describes potentialadvantages when using different carrier proteins in multivalentpneumococcal conjugate vaccines, but the PREVNAR™ product successfullyuses the same carrier for each of seven different serotypes.

A carrier protein may be covalently conjugated to a pneumococcalsaccharide directly or via a linker, as discussed above in relation tomeningococcal conjugates. Cross-linking conjugation techniques areparticularly useful for at least pneumococcal serotypes 4, 6B, 9V, 14,18C, 19F and 23F.

As discussed above for meningococcal saccharides, a pneumococcalsaccharide may comprise a full-length intact saccharide as prepared frompneumococcus, and/or may comprise fragments of full-length saccharides.Where more than one pneumococcal serotype is used then it is possible touse intact saccharides for each serotype, fragments for each serotype,or to use intact saccharides for some serotypes and fragments for otherserotypes. Where a composition includes saccharide from any of serotypes4, 6B, 9V, 14, 19F and 23F, these saccharides are preferably intact. Incontrast, where a composition includes serotype 18C saccharide it ispreferably depolymerised.

A serotype 3 saccharide may also be depolymerised, For instance, aserotype 3 saccharide can be subjected to acid hydrolysis fordepolymerisation [98] e.g. using acetic acid. The resulting fragmentsmay then be oxidised for activation (e.g. periodate oxidation, maybe inthe presence of bivalent cations e.g. with MgCl₂), conjugated to acarrier (e.g. CRM197) under reducing conditions (e.g. using sodiumcyanoborohydride), and then (optionally) any unreacted aldehydes in thesaccharide can be capped (e.g. using sodium borohydride) [98].Conjugation may be performed on lyophilized material e.g. afterco-lyophilizing activated saccharide and carrier.

A serotype 1 saccharide may be at least partially de-O-acetylated e.g.achieved by alkaline pH buffer treatment [99] such as by using abicarbonate/carbonate buffer. Such (partially) de-O-acetylatedsaccharides can be oxidised for activation (e.g. periodate oxidation),conjugated to a carrier (e.g. CRM197) under reducing conditions (e.g.using sodium cyanoborohydride), and then (optionally) any unreactedaldehydes in the saccharide can be capped (e.g. using sodiumborohydride) [99]. Conjugation may be performed on lyophilized materiale.g. after co-lyophilizing activated saccharide and carrier.

A serotype 19A saccharide may be oxidised for activation (e.g. periodateoxidation), conjugated to a carrier (e.g. CRM197) in DMSO under reducingconditions, and then (optionally) any unreacted aldehydes in thesaccharide can be capped (e.g. using sodium borohydride) [103].Conjugation may be performed on lyophilized material e.g. afterco-lyophilizing activated saccharide and carrier.

Pneumococcal conjugates can ideally elicit anticapsular antibodies thatbind to the relevant saccharide e.g. elicit an anti-saccharide antibodylevel ≧0.20 μg/mL [104]. The antibodies may be evaluated by enzymeimmunoassay (EIA) and/or measurement of opsonophagocytic activity (OPA).The EIA method has been extensively validated and there is a linkbetween antibody concentration and vaccine efficacy.

Further Antigens from Other Pathogen(s)

Compositions of the invention can include antigen(s) from furtherpathogen(s). The use of an aluminium hydroxyphosphate adjuvant, andavoidance of an aluminium hydroxide adjuvant, is advantageous in thecontext of such combinations because, as described above, the additionalantigens (in particular bacterial capsular saccharides) may be sensitiveto the hydroxide salt.

For example, the composition may comprise one or more of the followingfurther antigen(s):

-   -   an antigen from hepatitis B virus, such as the surface antigen        HBsAg.    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous haemagglutinin (FHA) from B.        pertussis, optionally also in combination with pertactin and/or        agglutinogens 2 and 3.    -   a diphtheria antigen, such as a diphtheria toxoid.    -   a tetanus antigen, such as a tetanus toxoid.    -   a saccharide antigen from Haemophilus influenzae B (Hib),        typically conjugated.    -   inactivated poliovirus antigen(s).

Where a diphtheria antigen is included in the composition it ispreferred also to include tetanus antigen and pertussis antigens.Similarly, where a tetanus antigen is included it is preferred also toinclude diphtheria and pertussis antigens. Similarly, where a pertussisantigen is included it is preferred also to include diphtheria andtetanus antigens. DTP combinations are thus preferred.

Extemporaneous Preparation

The invention also provides a kit comprising: (i) a first componentcomprising at least one fHBP antigen adsorbed to an aluminiumhydroxyphosphate adjuvant, as described above; and (ii) a secondcomponent comprising a non-meningococcal immunogen. The kit componentscan be mixed to give an immunogenic composition for administering to apatient to protect against multiple pathogens.

The invention also provides a method for preparing a combined vaccine,comprising a step of mixing: (i) a first component comprising at leastone fHBP antigen adsorbed to an aluminium hydroxyphosphate adjuvant, asdescribed above; and (ii) a second component comprising anon-meningococcal immunogen. The mixed material may then be administeredto a patient. The second component may be lyophilised, such that anaqueous first component reconstitutes it.

Pharmaceutical Compositions

The invention is concerned with immunogenic compositions foradministration to a patient. These compositions are pharmaceuticallyacceptable and will typically include a suitable carrier. A thoroughdiscussion of pharmaceutically acceptable carriers is available inreference 105.

Effective dosage volumes can be routinely established, but a typicalhuman dose of the composition has a volume of about 0.5 ml.

The pH of a composition of the invention is usually between 6 and 8, andmore preferably between 6.5 and 7.5 (e.g. about 7). As already discussedabove, compositions may include a buffer e.g. a Tris buffer, a citratebuffer, phosphate buffer, a succinate buffer (such as a sodium succinatebuffer), or a histidine buffer.

In the first aspect of the invention, a particular pH is used prior toand/or during adsorption, as explained above. If the adsorption isstable, however, that pH does not have to be maintained after adsorptionbut can be allowed to rise e.g. closer to neutral. After adsorption,therefore, such a composition may be buffered at a pH above theadjuvant's PZC.

Similarly, the pH of a composition according to the second aspect shouldbe in the range of 5.0 to 7.0 before and/or during adsorption, but maybe outside this range (e.g. in the range 7.0 to 8.0) after adsorption.Ideally, though, compositions of the second aspect are maintained with apost-adsorption pH in the range of 5.0 to 7.0 by the use of a buffer.

If adsorption has taken place at a pH above the adjuvant's PZC then, ifthe adsorption is stable, the pH does not have to be maintained but canbe allowed to drop e.g. closer to neutral. After adsorption, therefore,such a composition may be buffered at a pH below the adjuvant's PZC.

The pH of a composition according to the third aspect is within 1.2 pHunits of the adjuvant's PZC before and/or during adsorption, but may beoutside this range after adsorption. Ideally, though, compositions ofthe third aspect are maintained with a post-adsorption pH within 1.2 pHunits of the adjuvant's PZC.

In some embodiments, a composition of the invention includes a bufferwith a pKa between 3.5 and 6.5, particularly when used in combinationwith saline. This formulation is said to be useful with fHBP inreference 106. A succinate buffer with 1-10 mM succinate (e.g. 5 mM) isuseful, with a pH between 5.8 and 6.0. The composition may includeMgCl₂, KCl and/or NaCl.

The composition may be sterile and/or pyrogen-free. Compositions of theinvention may be isotonic with respect to humans.

Compositions of the invention may include sodium salts (e.g. sodiumchloride) to give tonicity. A concentration of 10±2 mg/ml NaCl istypical e.g. about 9 mg/ml.

Compositions of the invention for administration to patients areimmunogenic, and are more preferably vaccine compositions. Vaccinesaccording to the invention may either be prophylactic (i.e. to preventinfection) or therapeutic (i.e. to treat infection), but will typicallybe prophylactic. Immunogenic compositions used as vaccines comprise animmunologically effective amount of antigen(s), as well as any othercomponents, as needed. By ‘immunologically effective amount’, it ismeant that the administration of that amount to an individual, either ina single dose or as part of a series, is effective for treatment orprevention. This amount varies depending upon the health and physicalcondition of the individual to be treated, age, the taxonomic group ofindividual to be treated (e.g. non-human primate, primate, etc.), thecapacity of the individual's immune system to synthesise antibodies, thedegree of protection desired, the formulation of the vaccine, thetreating doctor's assessment of the medical situation, and otherrelevant factors. It is expected that the amount will fall in arelatively broad range that can be determined through routine trials.The antigen content of compositions of the invention will generally beexpressed in terms of the amount of protein per dose.

Meningococci affect various areas of the body and so the compositions ofthe invention may be prepared in various liquid forms. For example, thecompositions may be prepared as injectables, either as solutions orsuspensions. The composition may be prepared for pulmonaryadministration e.g. by an inhaler, using a fine spray. The compositionmay be prepared for nasal, aural or ocular administration e.g. as sprayor drops. Injectables for intramuscular administration are most typical.

Compositions of the invention may include an antimicrobial, particularlywhen packaged in multiple dose format. Antimicrobials such as thiomersaland 2-phenoxyethanol are commonly found in vaccines, but it is preferredto use either a mercury-free preservative or no preservative at all.

Compositions of the invention may comprise detergent e.g. a Tween(polysorbate), such as Tween 80. Detergents are generally present at lowlevels e.g. <0.01%, but higher levels have been suggested forstabilising antigen formulations [106] e.g. up to 10%. An examplecomposition may include from 0.01 to 0.05% polysorbate, and this isparticularly useful when using lipidated fHBP antigen(s).

Methods of Treatment

The invention also provides a method for raising an immune response in amammal, comprising administering a composition of the invention to themammal. The immune response is preferably protective againstmeningococcus and preferably involves antibodies. The method may raise abooster response in a patient that has already been primed.

The mammal is preferably a human. Where the vaccine is for prophylacticuse, the human is preferably a child (e.g. a toddler or infant) or ateenager, where the vaccine is for therapeutic use, the human ispreferably an adult. A vaccine intended for children may also beadministered to adults e.g. to assess safety, dosage, immunogenicity,etc.

The invention also provides compositions of the invention for use as amedicament. The medicament is preferably used, as described above, toraise an immune response in a mammal (i.e. it is an immunogeniccomposition) and is more preferably a vaccine.

The invention also provides the use of at least one fHBP antigen and analuminium hydroxyphosphate adjuvant in the manufacture of a medicamentfor raising an immune response, as described above, in a mammal.

These uses and methods are preferably for the prevention and/ortreatment of a disease caused by N. meningitidis e.g. bacterial (or,more specifically, meningococcal) meningitis, or septicemia.

One way of checking efficacy of therapeutic treatment involvesmonitoring meningococcal infection after administration of thecomposition of the invention. One way of checking efficacy ofprophylactic treatment involves monitoring immune responses againstantigens after administration of the composition. Immunogenicity ofcompositions of the invention can be determined by administering them totest subjects (e.g. children 12-16 months age, or animal models) andthen determining standard parameters including serum bactericidalantibodies (SBA) and ELISA titres (GMT) for meningococcus. These immuneresponses will generally be determined around 4 weeks afteradministration of the composition, and compared to values determinedbefore administration of the composition. A SBA increase of at least4-fold or 8-fold is preferred. Where more than one dose of thecomposition is administered, more than one post-administrationdetermination may be made.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by any other suitableroute. The invention may be used to elicit systemic and/or mucosalimmunity. Intramuscular administration to the thigh or the upper arm ispreferred. Injection may be via a needle (e.g. a hypodermic needle), butneedle-free injection may alternatively be used. A typical intramusculardose is 0.5 ml.

Dosage treatment can be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunisation scheduleand/or in a booster immunisation schedule. A primary dose schedule maybe followed by a booster dose schedule. Suitable timing between primingdoses (e.g. between 4-16 weeks), and between priming and boosting, canbe routinely determined.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., references107-113, etc.

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

Where the invention concerns an “epitope”, this epitope may be a B-cellepitope and/or a T-cell epitope, but will usually be a B-cell epitope.Such epitopes can be identified empirically (e.g. using PEPSCAN[114,115] or similar methods), or they can be predicted (e.g. using theJameson-Wolf antigenic index [116], matrix-based approaches [117],MAPITOPE [118], TEPITOPE [119,120], neural networks [121], OptiMer &EpiMer [122,123], ADEPT [124], Tsites [125], hydrophilicity [126],antigenic index [127] or the methods disclosed in references 128-132,etc.). Epitopes are the parts of an antigen that are recognised by andbind to the antigen binding sites of antibodies or T-cell receptors, andthey may also be referred to as “antigenic determinants”.

Where the invention uses a “purified” antigen, this antigen is separatedfrom its naturally occurring environment. For example, the antigen willbe substantially free from other meningococcal components, other thanfrom any other purified antigens that are present. A mixture of purifiedantigens will typically be prepared by purifying each antigen separatelyand then re-combining them, even if the two antigens are naturallypresent in admixture.

References to a percentage sequence identity between two amino acidsequences means that, when aligned, that percentage of amino acids arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 of ref.133. A preferred alignment is determined by the Smith-Waterman homologysearch algorithm using an affine gap search with a gap open penalty of12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is disclosed in ref. 134.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

MODES FOR CARRYING OUT THE INVENTION

Aluminium Adjuvants

Adsorption of fHBP to different aluminium adjuvants under differentconditions was studied. Various fHBP antigens were used, including asingle fHBP (predicted pI of 7.4) or hybrid mixtures of 2 or 3 fHBPs.Some experiments included additional non-fHBP meningococcal antigens.

With an aluminium hydroxide adjuvant at pH 6.5±0.5, 100% adsorption offHBP was seen with all single and mixed antigens. Full adsorption wasalso seen at slightly higher pH in the presence of 10 mM histidinebuffer. The presence of additional meningococcal polypeptide adjuvantsdid not reduce the degree of fHBP adsorption.

In contrast, with an aluminium hydroxyphosphate adjuvant at pH 7.0 thefHBP antigen was seen to be only 50% adsorbed. This pH is below theantigen's predicted pI and above the adjuvant's PZC.

Aluminium hydroxide adjuvants generally have a PZC of about 11.4. Thusneutral pH is below the adjuvant's PZC. In contrast, neutral pH is abovePZC of the aluminium hydroxyphosphate adjuvant.

Adsorption of fHBP to an aluminium hydroxyphosphate adjuvant was studiedat various pH. The following data show pH and adsorption data obtained24 hours after formulation. These three formulations have the sameprotein concentration (50 μg/ml) and adjuvant concentration (0.5 mg/ml),but use a 10 mM sodium phosphate buffer at different pH:

pH % adsorption 7.0 ~50% 5.8 ~95% 3.5 not adsorbed

Thus ˜95% adsorption was achieved in a pH 5.8 composition. This pH isapproximately equal to the adjuvant's PZC (slightly higher) but is wellbelow the antigen's pI. In contrast, at an increased pH (1.2 pH unitshigher) or decreased pH (2.3 pH units lower) adsorption was poor.

A high level of adsorption could also be achieved by increasing theamount of adjuvant 4.5-fold.

The influence of buffer and pH was studied in further experiments using1 mg/ml adjuvant and 100 μg/ml antigen. Results were as follows:

Buffer pH % adsorption Sodium phosphate 7.1 ~80% 10 mM 5.9 ~95% 5.5 ~95%4 ~80% Sodium phosphate 6.9 ~85% 5 mM 6.1 ~95% 5.9 ~95% Histidine 7 ~96%5 mM 5.9 ~98% 5.2 ~95%

Thus high adsorption (≧95%) to aluminium hydroxyphosphate could beachieved by selecting an appropriate pH. Adsorption levels above 85%were here seen only when the pH was within 1.2 pH units of theadjuvant's PZC (in the relevant buffer).

As mentioned above, these studies were performed with a fHBP having a pIof 7.4. This fHBP is referred to hereafter as fHBP-v1. Further studieswere performed with fHBP from two more meningococcal strains. Thepredicted pI for fHBP-v2 is 5.8, and for fHBP-v3 it is 6.1. Furthermore,a fusion to combine all three of v1, v2 and v3 was studied. Each ofthese four proteins was formulated at 100 μg/ml with 0.222 mg/mladjuvant and 9 mg/ml NaCl. Three different formulation pH wereinvestigated, namely pH 5, pH 6 and pH 7. The degree of adsorption ofthe fHBP proteins to the aluminium hydroxyphosphate was then determined.Results were as follows:

Adsorption at pH pI 5 6 7 v1 7.4 20-40% 90-95% 40-60% v2 5.8 >95% >95%<25% v3 6.1 90-95% >95% 80-85% v1 + v2 + v3 — >95% >95% 85-90%

These results confirm that the v1/v2/v3 combination, which includes onefHBP with a pI of 5.8 and another with a pI of 6.1 (i.e. both between5.0 and 7.0), could achieve adsorption levels of ≧85% using an aluminiumhydroxyphosphate adjuvant with a PZC between 5.0 and 7.0. Moreover, thehighest levels of adsorption for this combination were seen when the pHwas within 1.2 pH units of the adjuvant's point of zero charge (i.e. atpH 5 or pH 6, rather than at pH 7).

High adsorption was seen except (i) for v1 and v2, when the pH was morethan 1.2 units higher than the adjuvant's PZC (ii) for v1, when the pHwas lower than the adjuvant's PZC and the fHBP's pI was outside therange of 5.0 to 7.0. The relatively low adsorption of the v2 fHBP at pH7 could be overcome by adding a second fHBP with a pI in the range of5.0 to 7.0.

Thus mixtures of multiple fHBP variants with different pI values cansuccessfully be formulated with high levels of adsorption withoutrequiring aluminium hydroxide.

Immunostimulatory Oligonucleotide+Polycationic Polymer Adjuvant

As an alternative to using an aluminium-based adjuvants the inventioncan use a particulate complex of an immunostimulatory oligonucleotideand a polycationic polymer, such as IC31.

The three polypeptides which make up the ‘5CVMB’ vaccine disclosed inreference 12 (see also ref. 135) were adjuvanted with aluminiumhydroxide and/or IC31. One of these three polypeptides includes a fHBPantigen.

In a first set of experiments, nine groups of mice received 10 μg ofantigens, 3 mg/ml of aluminium hydroxide and varying doses of IC31.Groups received the following nine compositions, with groups 7-9receiving the same antigens as 1-6 but differently formulated:

Antigen dose (μg) IC31 volume* (μl) Al—H (mg/ml) 5 10 0 3 6 10 100 0 710 0 3 9 10 100 0 *A standard IC31 suspension was used. 100 μl of thissuspension gave full-strength. Lower volumes gave lower strengths. Topreserve the volume for the lower-strength compositions, buffer wasadded up to 100 μl.

Sera from the mice were tested against a panel of meningococcal strainsfor bactericidal activity.

Bactericidal titers from experiment MP03 were as follows against sixdifferent strains, A to F:

A B C D E F 5 >65536 2048 4096 8192 256 32768 6 >65536 4096 >8192 81921024 >65536 7 >65536 2048 4096 4096 256 4096 9 32768 8192 >8192 >81924096 >65536

Thus the titers obtained with IC31 were usually better than thoseobtained with Al—H.

The ‘5CVMB’ vaccine was combined with a tetravalent mixture ofmeningococcal conjugates against serogroups A, C, W135 and Y. Themixture was adjuvanted with Al—H or IC31 (at high or low concentration).Bactericidal titers were as follows against a panel with one strain fromeach of serogroups A, C, W135 and Y:

A C W135 Y Un-immunised <16 <16 <16 <16 No adjuvant 1024 256 128 512IC31^(high) 32768 16384 4096 4096 IC31^(low) 16384 8192 1024 2048Al-hydroxide 16384 8192 1024 4096

Thus the best titers were seen with IC31.

In separate experiments a triple-fusion polypeptide containing threevariants of fHBP, in the order II-III-I (as disclosed in reference 27),was adjuvanted with aluminium hydroxide or IC31.

In a first set of experiments, six groups of mice received 20 μg ofantigen (with or without a purification tag), 3 mg/ml of aluminiumhydroxide and 100 μl of IC31. Groups received the following:

Antigen dose (μg) Antigen tag IC31 volume (μl) Al—H (mg/ml) 1 20 No 1000 2 20 Yes 100 0 5 20 No 0 3 6 20 Yes 0 3

Sera from the mice were tested against a panel of meningococcal strainsfor bactericidal activity.

Sera from experiment MP05 were again tested against a panel of strains(25 in total). 56% of strains in group 1 (IC31, no tag) had a titer≧1:1024 vs. only 36% of strains in group 5 (Al—H, no tag). Similarly,76% of strains in group 1 had a titer ≧1:128 while this titer wasobserved in only 64% of strains in group 5. Looking at the untaggedantigens, 84% of strains in group 2 (IC31) had a titer ≧1:128 vs. 76% ingroup 6 (Al—H). Thus higher bactericidal titers were achieved usingIC31.

Further immunogenicity experiments used the fHBP_(II-III-I) antigen incombination with the NadA and 287-953 antigens in experiment MP04, withthe same groupings and strain panel. Group 1 showed a bactericidal titer≧1:128 in 100% of strains, compared to only 84% in group 5. With a morestringent threshold of ≧1:1024, sera from group 1 were bactericidalagainst 88% of strains, compared to only 56% in group 5. Similar resultswere observed with tagged antigens, where 88% of group 2 had abactericidal titer of ≧1:128 compared to 80% in group 6. Again,therefore, better anti-meningococcus immune responses were obtained withIC31.

In similar experiments the combination of fHBP_(II-III-I), NadA and287-953 was adjuvanted with Al—H or IC31. These compositions werecompared with a composition comprising the 5CVMB vaccine including outermembrane vesicles, adjuvanted with Al—H. The IC31-adjuvanated vaccineprovided a higher % coverage across 12 tested strains than any othercomposition.

Thus IC31 is an effective adjuvant for fHBP.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES

-   [1] Masignani et al. (2003) J Exp Med 197:789-799.-   [2] Welsch et al. (2004) J Immunol 172:5605-15.-   [3] Hou et al. (2005) J Infect Dis 192(4):580-90.-   [4] WO03/063766.-   [5] Fletcher et al. (2004) Infect Immun 72:2088-2100.-   [6] Zhu et al. (2005) Infect Immun 73(10):6838-45.-   [7] Cantini et al. (2006) J. Biol. Chem. 281:7220-7227-   [8] WO2004/048404-   [9] WO03/020756.-   [10] Sturgess et al. (1999) Vaccine 17:1169-1178.-   [11] U.S. Pat. No. 7,404,960.-   [12] Giuliani et al. (2006) PNAS USA 103:10834-9.-   [13] Hem & HogenEsch (2007) Chapter 4 of Vaccine Adjuvants and    Delivery Systems (ed. Singh).-   [14] Burrell et al. (2001) Vaccine 19:275-81.-   [15] Methods in Moelcular Medicine, Vol. 42 (ed. O'Hagan) Vaccine    Adjuvants . . .-   [16] WO01/22992.-   [17] Bjellqvist et al. (1993) Electrophoresis 14:1023-31.-   [18] Gasteiger et al. (2005) Protein Identification and Analysis    Tools on the ExPASy Server in The Proteomics Protocols Handbook (ed.    John M. Walker), Humana Press (2005).-   [19] Krieg (2003) Nature Medicine 9:831-835.-   [20] McCluskie et al. (2002) FEMS Immunology and Medical    Microbiology 32:179-185.-   [21] WO98/40100.-   [22] U.S. Pat. No. 6,207,646.-   [23] U.S. Pat. No. 6,239,116.-   [24] U.S. Pat. No. 6,429,199.-   [25] Schellack et al. (2006) Vaccine 24:5461-72.-   [26] Lingnau et al. (2007) Expert Rev Vaccines 6:741-6.-   [27] WO2004/084938.-   [28] Kamath et al. (2008) Eur J Immunol 38:1247-56.-   [29] Riedl et al. (2008) Vaccine 26:3461-8.-   [30] Kritsch et al. (2005) J Chromatography B 822:263-70.-   [31] Lingnau et al. (2003) Vaccine 20:3498-508.-   [32] Tettelin et al. (2000) Science 287:1809-1815.-   [33] WO00/66741.-   [34] WO99/57280-   [35] Martin et al. (1997) J Exp Med 185(7):1173-83.-   [36] WO96/29412.-   [37] U.S. Pat. No. 5,698,438.-   [38] Perkins-Balding et al. (2003) Microbiology 149:3423-35.-   [39] WO01/55182.-   [40] WO01/38350.-   [41] WO00/23595.-   [42] Ram et al. (2003) J Biol Chem 278:50853-62.-   [43] WO2004/014417.-   [44] WO98/53851-   [45] U.S. Pat. No. 6,531,131-   [46] WO00/26384.-   [47] U.S. Pat. No. 6,645,503-   [48] WO03/070282.-   [49] WO94/08021-   [50] WO2004/015099.-   [51] WO2007/144316.-   [52] WO2007/144317.-   [53] WO03/080678.-   [54] Glode et al. (1979) J Infect Dis 139:52-56-   [55] WO94/05325; U.S. Pat. No. 5,425,946.-   [56] Arakere & Frasch (1991) Infect. Immun. 59:4349-4356.-   [57] Michon et al. (2000) Dev. Biol. 103:151-160.-   [58] Rubinstein & Stein (1998) J. Immunol. 141:4357-4362.-   [59] WO2005/033148-   [60] WO2007/000314.-   [61] Research Disclosure, 453077 (January 2002)-   [62] EP-A-0378881.-   [63] EP-A-0427347.-   [64] WO93/17712-   [65] WO94/03208.-   [66] WO98/58668.-   [67] EP-A-0471177.-   [68] WO91/01146-   [69] Falugi et al. (2001) Eur J Immunol 31:3816-3824.-   [70] Baraldo et al. (2004) Infect Immun 72(8):4884-7.-   [71] EP-A-0594610.-   [72] Ruan et al. (1990) J Immunol 145:3379-3384.-   [73] WO00/56360.-   [74] Kuo et al. (1995) Infect Immun 63:2706-13.-   [75] Michon et al. (1998) Vaccine. 16:1732-41.-   [76] WO02/091998.-   [77] WO01/72337-   [78] WO00/61761.-   [79] WO00/33882-   [80] WO2007/000341.-   [81] Bethell G. S. et al., J. Biol. Chem., 1979, 254, 2572-4-   [82] Hearn M. T. W., J. Chromatogr., 1981, 218, 509-18-   [83] WO2007/000343.-   [84] Mol. Immunol., 1985, 22, 907-919-   [85] EP-A-0208375-   [86] WO00/10599-   [87] Gever et al., Med. Microbiol. Immunol, 165: 171-288 (1979).-   [88] U.S. Pat. No. 4,057,685.-   [89] U.S. Pat. Nos. 4,673,574; 4,761,283; 4,808,700.-   [90] U.S. Pat. No. 4,459,286.-   [91] U.S. Pat. No. 5,204,098-   [92] U.S. Pat. No. 4,965,338-   [93] U.S. Pat. No. 4,663,160.-   [94] WO2007/000342.-   [95] WHO Technical Report Series No. 927, 2005. Pages 64-98.-   [96] US-2008/0102498.-   [97] US-2006/0228381.-   [98] US-2007/0231340.-   [99] US-2007/0184072.-   [100] US-2006/0228380.-   [101] WO2008/143709.-   [102] WO2007/071707-   [103] US-2007/0184071.-   [104] Jodar et al. (2003) Vaccine 21:3265-72.-   [105] Gennaro (2000) Remington: The Science and Practice of    Pharmacy. 20th edition, ISBN: 0683306472.-   [106] WO2007/127665.-   [107] Methods In Enzymology (S. Colowick and N. Kaplan, eds.,    Academic Press, Inc.)-   [108] Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir    and C. C. Blackwell, eds, 1986, Blackwell Scientific Publications)-   [109] Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual,    3rd edition (Cold Spring Harbor Laboratory Press).-   [110] Handbook of Surface and Colloidal Chemistry (Birdi, K. S. ed.,    CRC Press, 1997)-   [111] Ausubel et al. (eds) (2002) Short protocols in molecular    biology, 5th edition (Current Protocols).-   [112] Molecular Biology Techniques: An Intensive Laboratory Course,    (Ream et al., eds., 1998, Academic Press)-   [113] PCR (Introduction to Biotechniques Series), 2nd ed. (Newton &    Graham eds., 1997, Springer Verlag)-   [114] Geysen et al. (1984) PNAS USA 81:3998-4002.-   [115] Carter (1994) Methods Mol Biol 36:207-23.-   [116] Jameson, B A et al. 1988, CABIOS 4(1):181-186.-   [117] Raddrizzani & Hammer (2000) Brief Bioinform 1(2):179-89.-   [118] Bublil et al. (2007) Proteins 68(1):294-304.-   [119] De Lalla et al. (1999) J. Immunol. 163:1725-29.-   [120] Kwok et al. (2001) Trends Immunol 22:583-88.-   [121] Brusic et al. (1998) Bioinformatics 14(2):121-30-   [122] Meister et al. (1995) Vaccine 13(6):581-91.-   [123] Roberts et al. (1996) AIDS Res Hum Retroviruses 12(7):593-610.-   [124] Maksyutov & Zagrebelnaya (1993) Comput Appl Biosci 9(3):291-7.-   [125] Feller & de la Cruz (1991) Nature 349(6311):720-1.-   [126] Hopp (1993) Peptide Research 6:183-190.-   [127] Welling et al. (1985) FEBS Lett. 188:215-218.-   [128] Davenport et al. (1995) Immunogenetics 42:392-297.-   [129] Tsurui & Takahashi (2007) J Pharmacol Sci. 105(4):299-316.-   [130] Tong et al. (2007) Brief Bioinform. 8(2):96-108.-   [131] Schirle et al. (2001) J Immunol Methods. 257(1-2):1-16.-   [132] Chen et al. (2007) Amino Acids 33(3):423-8.-   [133] Current Protocols in Molecular Biology (F. M. Ausubel et al.,    eds., 1987) Supplement 30-   [134] Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489.-   [135] WO2004/032958.

1-22. (canceled) 23: An immunogenic composition comprising two different meningococcal factor H binding protein (fHBP) antigens, both of which are adsorbed to aluminium hydroxyphosphate adjuvant, wherein (i) both of the meningococcal fHBP antigens have an isoelectric point between 5.0 and 7.0, and (ii) the aluminium hydroxyphosphate adjuvant has a point of zero charge between 5.0 and 7.0, wherein the two different fHBP antigens are: (i) a first polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 4; and (ii) a second polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:
 6. 24: An immunogenic composition comprising two different meningococcal factor H binding protein (fHBP) antigens, both of which are adsorbed to aluminium hydroxyphosphate adjuvant, wherein both of the meningococcal fHBP antigens have an isoelectric point between 5.0 and 7.0, wherein the two different meningococcal fHBP antigens are: (a) a first polypeptide comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 4 and being lipidated at a N-terminus cysteine; and (b) a second polypeptide comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 6 and being lipidated at a N-terminus cysteine, and wherein the composition: (i) includes a histidine buffer to maintain a pH in the range of 5.0 to 7.0; (ii) has an Al⁺⁺⁺ concentration between 0.2 and 1 mg/ml; (iii) includes NaCl; (iv) includes polysorbate 80; and (v) includes between 1 and 500 μg/dose of each fHbp polypeptide. 25: An immunogenic composition comprising two different meningococcal factor H binding protein (fHBP) antigens, both of which are adsorbed to aluminium hydroxyphosphate adjuvant, wherein (i) both of the meningococcal fHBP antigens have an isoelectric point between 5.0 and 7.0, and (ii) the aluminium hydroxyphosphate adjuvant has a point of zero charge between 5.0 and 7.0, wherein at least 85% of the each of the fHBP antigens is adsorbed to the aluminium hydroxyphosphate adjuvant. 26: An immunogenic composition comprising two different meningococcal factor H binding protein (fHBP) antigens, both of which are adsorbed to aluminium hydroxyphosphate adjuvant, wherein both of the meningococcal fHBP antigens have an isoelectric point between 5.0 and 7.0, wherein the two different meningococcal fHBP antigens are: (a) a first polypeptide comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 4; and (b) a second polypeptide comprising an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 6, and wherein the composition: (i) includes a histidine buffer to maintain a pH in the range of 5.0 to 7.0; (ii) has an Al⁺⁺⁺ concentration <1 mg/ml; (iii) includes NaCl; (iv) includes polysorbate 80; and (v) includes between 1 and 500 μg/dose of each fHbp polypeptide. 27: A method for adsorbing two different meningococcal fHBP antigens to an aluminium hydroxyphosphate adjuvant to give an immunogenic composition, wherein (i) both of the meningococcal fHBP antigens have an isoelectric point between 5.0 and 7.0, (ii) the aluminium hydroxyphosphate adjuvant has a point of zero charge between 5.0 and 7.0, and (iii) adsorption of both of the fHBP antigens takes place at a pH between 5.0 and 7.0. 28: A method for adsorbing two different meningococcal fHBP antigens to an aluminium hydroxyphosphate adjuvant, wherein (i) the meningococcal fHBP antigens both have isoelectric points which are greater than the adjuvant's point of zero charge and (ii) adsorption of each antigen takes place at a buffered pH which is within 1.2 pH units of the adjuvant's point of zero charge. 29: A method for adsorbing two different meningococcal fHBP antigens to an aluminium hydroxyphosphate adjuvant, wherein adsorption of both of the fHBP antigens takes place at a pH which is equal to or below the aluminium hydroxyphosphate's point of zero charge. 30: A method for raising an immune response in a human comprising administering to the human a priming aqueous suspension composition comprising two different meningococcal factor H binding protein (fHBP) antigens, both of which are adsorbed to aluminium hydroxyphosphate adjuvant, wherein both of the meningococcal fHBP antigens have an isoelectric point between 5.0 and 7.0, wherein the two different meningococcal fHBP antigens are: (a) a first polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 4; and (b) a second polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 6, and wherein the priming aqueous suspension composition: (i) includes a histidine buffer to maintain a pH in the range of 5.0 to 7.0; (ii) has an Al⁺⁺⁺ concentration <1 mg/ml; (iii) includes polysorbate 80; and (iv) includes between 1 and 500 μg/dose of each fHbp polypeptide. 31: The method of claim 30, wherein the administration is by intramuscular injection. 32: The method of claim 31, wherein the intramuscular administration is to the upper arm. 33: The method of claim 30, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 4 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:
 6. 34: The method of claim 30, wherein the priming aqueous suspension composition has a volume of about 0.5 ml. 35: The method of claim 30, wherein the priming aqueous suspension composition includes about 60 μg/dose of each fHbp polypeptide. 36: The method of claim 30, wherein the histidine is at 10 mM concentration. 37: The method of claim 30, further comprising administering to the human a second aqueous suspension composition comprising two different meningococcal factor H binding protein (fHBP) antigens, both of which are adsorbed to aluminium hydroxyphosphate adjuvant, wherein both of the meningococcal fHBP antigens have an isoelectric point between 5.0 and 7.0, wherein the two different meningococcal fHBP antigens are: (a) a first polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 4; and (b) a second polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 6, and wherein the second aqueous suspension composition: (i) includes a histidine buffer to maintain a pH in the range of 5.0 to 7.0; (ii) has an Al⁺⁺⁺ concentration <1 mg/ml; (iii) includes polysorbate 80; and (iv) includes between 1 and 500 μg/dose of each fHbp polypeptide, wherein the boosting dose is administered between 4 and 16 weeks after administration of the priming aqueous suspension composition. 38: A method for increasing an immune response in a human comprising administering to the human a second aqueous suspension composition comprising two different meningococcal factor H binding protein (fHBP) antigens, both of which are adsorbed to aluminium hydroxyphosphate adjuvant, wherein both of the meningococcal fHBP antigens have an isoelectric point between 5.0 and 7.0, wherein the two different meningococcal fHBP antigens are: (a) a first polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 4; and (b) a second polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 6, and wherein the second aqueous suspension composition: (i) includes a histidine buffer to maintain a pH in the range of 5.0 to 7.0; (ii) has an Al⁺⁺⁺ concentration <1 mg/ml; (iii) includes polysorbate 80; and (iv) includes between 1 and 500 μg/dose of each fHbp polypeptide, wherein the boosting dose is administered between 4 and 16 weeks after the human was administered a priming aqueous suspension composition comprising two different meningococcal factor H binding protein (fHBP) antigens, both of which are adsorbed to aluminium hydroxyphosphate adjuvant, wherein both of the meningococcal fHBP antigens have an isoelectric point between 5.0 and 7.0, wherein the two different meningococcal fHBP antigens are: (a) a first polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 4; and (b) a second polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 6, and wherein the priming aqueous suspension composition: (i) includes a histidine buffer to maintain a pH in the range of 5.0 to 7.0; (ii) has an Al⁺⁺⁺ concentration <1 mg/ml; (iii) includes polysorbate 80; and (iv) includes between 1 and 500 μg/dose of each fHbp polypeptide. 39: The method of claim 38, further comprising administering to the human a third aqueous suspension composition comprising two different meningococcal factor H binding protein (fHBP) antigens, both of which are adsorbed to aluminium hydroxyphosphate adjuvant, wherein both of the meningococcal fHBP antigens have an isoelectric point between 5.0 and 7.0, wherein the two different meningococcal fHBP antigens are: (a) a first polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 4; and (b) a second polypeptide comprising an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 6, and wherein the third aqueous suspension composition: (i) includes a histidine buffer to maintain a pH in the range of 5.0 to 7.0; (ii) has an Al⁺⁺⁺ concentration <1 mg/ml; (iii) includes polysorbate 80; and (iv) includes between 1 and 500 μg/dose of each fHbp polypeptide, wherein the third aqueous suspension composition is administered after the second aqueous suspension composition. 40: The method of claim 39, wherein the human has an increase in serum bactericidal antibodies for meningococcus that is at least 8-fold higher four weeks after administration of the third aqueous suspension composition as compared to before administration of the first aqueous suspension composition. 